Compositions and methods for treating metabolic diseases

ABSTRACT

The disclosure provides compositions and methods of treating a metabolic disease, such as, e.g., diabetes and hyperlipidemia, in a subject, by administering to the subject a therapeutically effective amount of a polypeptide derived from hepatitis B virus or a pharmaceutical composition comprising the polypeptide.

This application is a divisional application filing under 35 U.S.C. §121 of U.S. application Ser. No. 16/305,790, filed on Nov. 29, 2018,which claims the priority to and benefits of Chinese Application No.201610370442.4, filed May 30, 2016, and International ApplicationPCT/CN2017/086558, filed on May 31, 2017. The content of theseapplications is incorporated herein by reference.

This application contains a Sequence Listing as an XML file entitled“13230.0001-01000 Sequence Listing” having a size of 94,823 bytes andcreated on Aug. 10, 2023. The information contained in the SequenceListing is incorporated by reference herein.

This disclosure relates to compositions and methods for treating ametabolic disease such as diabetes and hyperlipidemia. In certainembodiments, the disclosure relates to the treatment of a metabolicdisease with a polypeptide derived from hepatitis B virus.

Metabolic diseases are caused by an imbalance of metabolites includingcarbohydrates, fats, lipids, and minerals that are crucial forwell-being of a living organism. For example, type II diabetes andhyperlipidemia represent two most common metabolic diseases. Theimbalance of metabolites may occur as a result of various factorsincluding aging, behavior, genetics, and environmental influences, andoften multiple factors in combination contribute the manifestation ofthe disease. Patients suffering from metabolic diseases may display awide range of symptoms including, for example, hyperglycemia,hyperinsulinemia, hyperlipidemia, insulin resistance, and dysregulationof other metabolites such as amino acids and minerals. It is oftendifficult to identify the underlying cause of metabolic diseases, makingthem difficult to treat effectively. Furthermore, patients sufferingfrom metabolic diseases may have a risk of developing seriouscomplications associated with the diseases, such as hypertension,cardiovascular diseases, kidney damages, and nerve damages.

Due to the heterogeneity of these diseases, patients suffering frommetabolic diseases may require a number of different medications,targeting multiple metabolic pathways in an attempt to address multiplesymptoms simultaneously. For instance, one study showed that the type IIdiabetic patients surveyed in the study were taking an average of 8.4different drug compounds per day (see, e.g., Bauer et al, DiabeticMedicine, 31:1078-85 (2014)). One of the reasons that the patientssuffering from metabolic diseases have to take various differentmedicines is because those medicines are often specialized to address aparticular symptom or pathway of the metabolic disease and thus are notcapable of targeting other related symptoms. Unfortunately, takingmultiple medicines can affect the life quality of the patients andultimately worsen the course of disease progression. Indeed, asstatistics show, type II diabetic patients experience difficultyadhering to their medication regimens partially because of thecomplexity of these regimens (see, e.g., Garcia-Perez et al, DiabetesTherapy, 4:175-94 (2013)).

Furthermore, metabolic diseases often involves a complicated network ofsignaling pathways, and therefore targeting one particular pathway byone agent does not always lead to a therapeutically relevant effect inpatients. For instance, cyclosporine A (CsA), an immunosuppressant drugwidely used in organ transplantation to prevent rejection, has beenshown to inhibit bile acid uptake and HBV entry into culturedhepatocytes mediated sodium taurocholate cotransporting polypeptide(NTCP), which is a Na⁺-dependent bile acid transporter that transportsbile acids from blood stream to hepatocytes (Watashi et al, Hpatology,59:1726-37 (2014)). However, treatment with CsA can produce deleteriouseffects on glucose metabolism and impair insulin response (Dresner etal, Surgery, 106(2):163-69 (1989)). In addition, CsA can inducehyperlipidemia in patients by increasing the total cholesterol level,primarily due to an increase in the low-density lipoprotein (LDL)cholesterol level (Ballantyne et al., JAMA, 262(1):53-56 (1989)).Various other compounds have been shown to bind to NTCP, but they do notproduce a uniform effect on NTCP as some of those compounds function asan inhibitor while others function as an enhancer (Kim et al, J.Pharmacol. Exp. Ther., 291(3):1204-09 (1999)). While most of those drugshave not been validated for their therapeutic outcomes in treatingmetabolic diseases to date, some enhancers of NTCP produce oppositeeffects on glucose or lipid metabolism (see, e.g., Beaudoin et al.,Appl. Physiol. Nutr. Metab. 38(2):140-47 (2013); Thelle et al., N. Engl.J. Med. 308(24):1454-57 (1983); Phillips et al., Br. Med. J. 292:1319-21(1986); Boden et al., Circulation, 85(6):2039-44 (1992)). It is alsounclear whether regulating NTCP in vivo would result in anytherapeutically relevant effect, because subjects with NTCP deficiencydid not exhibit any clear clinical phenotype (see Vaz et al, Hepatology,61(1):260-267 (2015)). Thus, there is an urgent need to develop a newmedication for metabolic diseases, potentially capable of addressingmultiple symptoms simultaneously with a potent therapeutic efficacy.

HBV viral envelope contains three surface antigen proteins: large (L),medium (M), and small (S). These proteins are coded by a single openreading frame on the S gene, starting from three different translationinitiating sites, i.e., L (Pre-S1+Pre-S2+S), M (Pre-S2+S), and S (S).The HBV is divided into four major serotypes (adr, adw, ayr, ayw) basedon antigenic epitopes present on its envelope proteins, and into eightgenotypes (A-H) according to overall nucleotide sequence variation ofthe genome. During viral infection, the Pre-S1 region on the L proteinof HBV was shown to bind to NTCP (Yan et al, eLife, 1:e00049 (2012)).

This disclosure provides compositions and methods for treating ametabolic disease with a polypeptide derived from HBV. In someembodiments, the polypeptides described herein include polypeptidesderived from the pre-S1 region of any one of HBV genotypes A, B, C, D,E, F, G, and H. The disclosure further provides HBV-derived polypeptidesthat are capable of altering metabolism such as glucose and lipidmetabolism in a subject, including humans as well as pharmaceuticallyrelevant animal models.

In some aspects, the disclosure provides a pharmaceutical compositioncomprising a polypeptide described herein, wherein when administered toa subject in need thereof, the pharmaceutical composition provides serumconcentrations of the polypeptide that allow for bidirectionalregulation of NTCP-mediated bile acid uptake in the subject.

In some aspects, the disclosure provides methods of treating a metabolicdisease in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the polypeptide describedherein or a pharmaceutical composition comprising the polypeptide suchthat the serum concentrations of the administered polypeptide allow forbidirectional regulation of NTCP-mediated bile acid uptake in thesubject, wherein the polypeptide comprises an amino acid sequencederived from Hepatitis B virus (HBV).

In some aspects, the disclosure relates to methods of lowering a serumlipid level in a subject in need thereof by administering to the subjecta therapeutically effective amount of the polypeptide described hereinor of a pharmaceutical composition comprising the polypeptide such thatthe serum concentrations of the administered polypeptide allow forbidirectional regulation of NTCP-mediated bile acid uptake in thesubject. In some embodiments, the serum lipid may include, e.g., totalcholesterol (“TC”), triglyceride (“TG”), and LDL-C.

In certain aspects, the disclosure also relates to methods of lowering ablood glucose level in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of thepolypeptide described herein or a pharmaceutical composition comprisingsuch polypeptide.

In some embodiments, when the serum concentration of the administeredpolypeptide in the subject is at or below 93 nmol/L, the polypeptideenhances NTCP-mediated bile acid uptake in the subject. In someembodiments, when the serum concentration of the administeredpolypeptide in the subject is above 93 nmol/L, the polypeptide inhibitsNTCP-mediated bile acid uptake in the subject. In some embodiments, theserum concentration of the polypeptide in the subject reaches a peakconcentration (i.e., C_(max)) at about 20 minutes after theadministration. Thus, in some embodiments, T_(max) of the polypeptidedescribed herein is about 20 minutes. In some embodiments, the peakconcentration is more than 93 nmol/L.

In some embodiments, a subject administered with the polypeptidedescribed herein suffers from or is at risk of developing a metabolicdisease. In some embodiments, the metabolic disease involvesdysregulation of lipid metabolism. In some embodiments, the metabolicdisease is a cholesterol-related disorder. In some embodiments, themetabolic disease is hyperlipidemia (e.g., hypertriglyceridemia,hypercholesterolemia, or a combination thereof). In some embodiments,the metabolic disease involves dysregulation of glucose metabolism. Insome embodiments, the metabolic disease is hyperglycemia. In someembodiments, the metabolic disease is diabetes or obesity. In someembodiments, the subject suffers from or is at risk of developingcardiovascular diseases (e.g., atherosclerotic diseases), heartdiseases, or kidney impairment.

In some embodiments, the polypeptide described herein is capable ofreducing or stabilizing the level or activity of one or more chemical orbiological molecules associated with metabolism in the subject. Thechemical or biological molecule associated with metabolism is chosenfrom glucose, cholesterol, triglyceride, free fatty acids, amino acids,hormones, LDL-C, HDL-C, HbA1c, blood urea nitrogen, and minerals. Insome embodiments, the polypeptide described herein is also capable ofreducing or stabilizing the level or value of one or more physiologicalparameters that measure metabolic changes. The physiological parameteris chosen from glycemia, blood pressure, body weight, fat mass, bodymass index (BMI), inflammation, atherosclerosis index, heart index,kidney index, total fat index, and homeostatic model assessment (HOMA)index.

In some embodiments, the polypeptide described herein comprises an aminoacid sequence derived from the pre-S1 region of HBV genotype A, B, C, D,E, F, G, or H. In certain embodiments, the polypeptide described hereincomprises the sequence of amino acids 13-59 of the pre-S1 region of HBVgenotype C. In additional embodiments, the polypeptide described hereincomprises an amino acid sequence derived from the pre-S1 region of anyother HBV genotype that corresponds to amino acids 13-59 of the pre-S1region of HBV genotype C. In some embodiments, the polypeptide comprisesan amino acid sequence selected from SEQ ID NOs: 21-40.

In some embodiments, one or more amino acid residues of the polypeptidedescribed herein are deleted, substituted, or inserted while maintainingthe ability to bind to NTCP and bidirectionally regulate NTCP-mediatedtransport of bile acid into hepatocytes. In certain embodiments, thepolypeptide described herein comprises a native flanking amino acidsequence from the pre-S1 region of HBV genotype A, B, C, D, E, F, G, orH. In other embodiments, the polypeptide described herein has at leastabout 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity to any one of the amino acid sequences selected from SEQ IDNOs: 21-40. In some embodiments, the polypeptide comprises the glycinecorresponding to amino acid 13 of the pre-S1 region of HBV genotype Cand/or the asparagine corresponding to amino acid 20 of the pre-S1region of HBV genotype C.

In some embodiments, the polypeptide described herein comprises anN-terminal modification with a hydrophobic group and/or a C-terminalmodification that is capable of stabilizing the polypeptide. Thehydrophobic group may be chosen from, e.g., myristic acid, palmiticacid, stearic acid, oleic acid, linoleic acid, cholesterol, andarachidonic acid. The C-terminal modification may be chosen from, e.g.,amidation (amination), isopentanediolization, and any C-terminalmodification that is capable of stabilizing the polypeptide. In certainembodiments, the polypeptide described herein comprises an N-terminalmodification with myristic acid and/or a C-terminal modification withamination. In some embodiments, the polypeptide described hereincomprises an amino acid sequence chosen from SEQ ID NOs: 21-40. In someembodiments, the polypeptide described herein comprises the amino acidsequence of SEQ ID NO: 23.

In one aspect, the polypeptide described herein is capable of reducingone or more symptoms associated with the metabolic disease. In someembodiments, the polypeptide described herein or the pharmaceuticalcomposition comprising such polypeptide is administered to the subjectbefore, concurrently with, or after the administration of atherapeutically effective amount of at least one a second agent. Thesecond agent may be chosen from, e.g., an antihyperlipidemic agent, anantihyperglycemic agent, an antidiabetic agent, an antiobesity agent,and a bile acid analogue. For example, the second agent may be chosenfrom, e.g., insulin, metformin, sitagliptin, colesevelam, glipizide,simvastatin, atorvastatin, ezetimibe, fenofibrate, nicotinic acid,orlistat, lorcaserin, phentermine, topiramate, obeticholic acid, andursodeoxycholic acid.

In some embodiments, the polypeptide described herein or thepharmaceutical composition comprising such polypeptide is administeredto the subject by at least one mode including, e.g., parenteral,intrapulmonary, intranasal, intralesional, intramuscular, intravenous,intraarterial, intraperitoneal, and subcutaneous administration. In someembodiments, the polypeptide described herein or the pharmaceuticalcomposition comprising such polypeptide is administered to the subjectsubcutaneously.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows an exemplary graph depicting the purity of Cmyr-47 asmeasured by high pressure liquid chromatography (HPLC). FIG. 1B shows anexemplary graph depicting the molecular weight of Cmyr-47 as confirmedby Mass Spectrometry.

FIG. 2A shows that Cmyr-47 labeled with FIFC binds to tupaia primaryhepatocytes.

FIG. 2B shows that Cmyr-47 labeled with FIFC binds to HepG2 cells, ahuman hepatocyte derived cell line.

FIG. 3A shows that Cmyr-47 labeled with FIFC does not bind to controlL02 cells (“BLANK-L02”). FIG. 3B shows that Cmyr-47 labeled with FIFCbinds to NTCP expressing L02 cells (“NTCP-L02”).

FIG. 4 shows that Cmyr-47 labeled with FIFC binds to NTCP expressingHEK293 cells (“NTCP-293”) but does to bind to control HEK293 cells(“BLANK-293”). A polypeptide derived from heron HBV, labeled with FIFC,was used as a control polypeptide.

FIG. 5A shows the effect of Cmyr-47 on bile acids uptake in vitro.Cyclosporine A (“CsA”) was used as a positive control. FIG. 5Billustrates the bidirectional effect of Cmyr-47 on bile acids uptake.FIGS. 5C and 5D show the effect of CsA on bile acids uptake in vitro andconfirm the inhibitory effect of CsA. FIGS. 5E and 5F show the effect ofHBV-derived polypeptides on bile acids uptake in vitro at a lowconcentration molarly equivalent to 62.5 ng/ml (11.58 nmol/L) Cmyr-47and at a high concentration molarly equivalent to 1 μg/ml (185.23nmol/L) Cmyr-47, respectively.

FIG. 6 shows the changes of serum total cholesterol (“TC”) inhyperlipidemic golden hamsters treated with Cmyr-47 or CsA for 4 weeks.Golden hamsters fed with regular diet were treated with PBS and used asa “Normal Control” while hyperlipidemic golden hamsters treated with PBSwere used as a “Model Control.” Hyperlipidemic golden hamsters treatedwith Fenofibrate (“Positive Treatment”) were used as a positive control.

FIG. 7A shows the level of serum TC prior to treatments withpolypeptides derived from HBV. FIG. 7B shows the level of serum TC after4 weeks of the treatments.

FIG. 8 depicts the changes of serum triglycerides (“TG”) ofhyperlipidemic golden hamsters during 4 weeks of Cmyr-47 treatment.

FIG. 9A shows the level of serum TG prior to treatments withpolypeptides derived from HBV. FIG. 9B shows the level of serum TG after4 weeks of the treatments.

FIG. 10 depicts the level of serum LDL-C of hyperlipidemic goldenhamsters after 4 weeks of Cmyr-47 treatment.

FIG. 11A shows the level of serum LDL-C before being treated withpolypeptides derived from HBV. FIG. 11B shows the level of serum LDL-Cafter 4 weeks of the treatments.

FIG. 12 shows the level of serum HDL-C of hyperlipidemic golden hamstersafter 4 weeks of Cmyr-47 treatment.

FIG. 13 depicts atherosclerosis index (“AI”) of hyperlipidemic goldenhamsters after 4 weeks of Cmyr-47 treatment.

FIG. 14 shows the changes of serum total bile acids (TBA) ofhyperlipidemic golden hamsters after 4 weeks of Cmyr-47 treatment.Fenofibrate and CsA were also tested for comparison.

FIG. 15 shows the changes of serum TC of hyperlipidemic golden hamsterstreated with three different doses (1 mg/kg, 3 mg/kg, and 10 mg/kg) ofCmyr-47 for 4 weeks. Golden hamsters fed with regular diet were treatedwith PBS and used as a “Normal Control,” while hyperlipidemic goldenhamsters treated with PBS were used as a “Model Control.”

FIG. 16 shows the changes of serum TG of hyperlipidemic golden hamsterstreated with 1 mg/kg, 3 mg/kg, or 10 mg/kg of Cmyr-47 for 4 weeks.

FIG. 17 shows the changes of serum glucose (“GLU”) in Zucker diabeticfatty rats during 4 weeks of Cmyr-47 or CsA treatment. Zucker lean ratstreated with PBS were used as a “Normal Control” while Zucker diabeticfatty rats treated with PBS were used as a “Model Control.” Zuckerdiabetic fatty rats treated with metformin (“Positive Treatment”) wereused as a positive control.

FIG. 18A shows the level of serum GLU before being treated withpolypeptides derived from HBV. FIG. 18B shows the level of serum GLUafter 4 weeks of the treatments.

FIG. 19 shows the changes of HbA1c of Zucker diabetic fatty rats during4 weeks of Cmyr-47 treatment.

FIG. 20A shows the level of HbA1c prior to treatments with polypeptidesderived from HBV. FIG. 20B shows the level of HbA1c after 4 weeks of thetreatments.

FIG. 21 shows the changes of insulin in Zucker diabetic fatty ratsduring 4 weeks of Cmyr-47 treatment.

FIG. 22 shows the changes of serum TC in Zucker diabetic fatty ratsduring 4 weeks of Cmyr-47 treatment.

FIG. 23A shows the level of serum TC prior to treatments withpolypeptides derived from HBV. FIG. 23B shows the level of serum TCafter 4 weeks of the treatments.

FIG. 24 shows the changes of serum TG in Zucker diabetic fatty ratsduring 4 weeks of Cmyr-47 treatment.

FIG. 25A shows the level of serum TG prior to treatments withpolypeptides derived from HBV. FIG. 25B shows the level of serum TGafter 4 weeks of the treatments.

FIG. 26 shows the level of blood urea nitrogen (“BUN”) in Zuckerdiabetic fatty rats after 4 weeks of Cmyr-47 treatment.

FIGS. 27A-C depict heart index, kidney index, and total fat index ofZucker diabetic fatty rats after 4 weeks of Cmyr-47 treatment.“Cmyr-47(L)” indicates the dose of 10 mg/kg/d of Cmyr-47 while“Cmyr-47(Hi)” indicates the dose of 30 mg/kg/d of Cmyr-47.

FIG. 28 shows the level of serum TBA in Zucker diabetic fatty rats after4 weeks of Cmyr-47 treatment.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. For the purposes of thepresent disclosure, the following terms are defined below.

The articles “a” and “an” refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. For example, “anelement” means one element or more than one element.

The term “or” means, and is used interchangeably with, the term“and/or,” unless context clearly indicates otherwise.

To the extent that the term “contain,” “include,” “have,” or grammaticalvariants of such term are used in either the disclosure or the claims,such term is inclusive in a manner similar to the term “comprising” as“comprising” is interpreted when employed as a transitional word in aclaim. The term “including” or its grammatical variants mean, and areused interchangeably with, the phrase “including but not limited to.”

The term “about” means a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight, or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. When the term “about” is used in conjunctionwith a numerical range, it modifies that range by extending theboundaries above and below the numerical values set forth. In general,the term “about” is intended to modify a numerical value above and belowthe stated value by a variance of 510%.

I. Polypeptides

Certain aspects of the present disclosure provide polypeptides derivedfrom HBV for treating a metabolic disease including, e.g., diabetes andhyperlipidemia. The polypeptides may be derived from the pre-S1 regionof HBV and may be capable of binding to NTCP in vitro, such as, e.g., ina solution or a cell-free system (e.g., a cell lysate or in areconstituted system), or in a cell, such as, e.g., ex vivo in a cell inculture (e.g., a cell expressing NTCP, or a hepatocyte), or in vivo in acell within a subject. The subject may be a mammal. In some embodiments,the subject may be a human.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably and encompass full-length proteins and fragments, aswell as variants of the full-length proteins and the fragments. Suchfragments and variants of the polypeptide described herein retain atleast the biological activities of the polypeptide to bind to NTCP andbidirectionally regulate NTCP-mediated transport of bile acid intohepatocytes. The “polypeptide,” “peptide,” and “protein” can includenatural and/or non-natural amino acid residues. Those terms also includepost-translationally modified proteins, including, e.g., glycosylated,sialylated, acetylated, and/or phosphorylated proteins. The terms alsoinclude chemically modified proteins at one or more amino acid residues,such as, e.g., at the N-terminus and/or at the C-terminus. For instance,the N-terminus of the polypeptide disclosed herein can be modified by ahydrophobic group such as, e.g., myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, cholesterol, and arachidonic acid. Insome embodiments, the C-terminus of the polypeptide disclosed herein canbe modified to stabilize the polypeptide. The C-terminus modificationmay be chosen from amidation (amination), isopentanediolization, and anyother C-terminal modification capable of stabilizing the polypeptide.

As used herein, the term “polypeptide derived from HBV” or “HBV-derivedpolypeptide” refers to the origin or source of the polypeptide as beingfrom HBV, and may include native, recombinant, synthesized, or purifiedpolypeptides. The term “polypeptide derived from HBV” or “HBV-derivedpolypeptide” refers to a full-length native HBV polypeptide or fragmentsthereof, as well as variants of the full-length native polypeptide orits fragments. In some embodiments, the fragment may consist of at least3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids,at least 20-30 amino acids, at least 30-50 amino acids, or the entireamino acids of the native sequence, or may be otherwise identifiable toone of ordinary skill in the art as having its origin in the nativesequence. In some embodiments, the polypeptide described herein may bederived from the pre-S1 region of the L protein of any HBV subtype. Insome embodiment, the polypeptide described herein may comprise theentire pre-S1 region of the L protein of any HBV subtype. In certainembodiments, the polypeptide described herein may be derived from thepre-S1 region of the L protein of any one of HBV genotypes A, B, C, D,E, F, G, and H. The genomic sequences of these HBV genotypes can befound in GenBank Accession Nos. KC875260 (SEQ ID NO: 41), AY220704 (SEQID NO: 42), AF461363 (SEQ ID NO: 43), AY796030 (SEQ ID NO: 44), AB205129(SEQ ID NO: 45), DQ823095 (SEQ ID NO: 46), HE981176 (SEQ ID NO: 47), andAB179747 (SEQ ID NO: 48), respectively. In certain embodiments, thepolypeptide described herein may be derived from the pre-S1 region ofthe L protein of HBV genotype C. The polypeptide derived from HBVdescribed herein retains one or more biological activities describedherein of the corresponding native HBV polypeptide, including at leastthe biological activities of the polypeptide to bind to NTCP andbidirectionally regulate NTCP-mediated transport of bile acid intohepatocytes.

“Variant” as used herein in connection with the polypeptide describedherein, a polypeptide derived from HBV, or an HBV-derived polypeptidemeans a polypeptide that differs from a given polypeptide (i.e., thepolypeptide described herein, the polypeptide derived from HBV, or theHBV-derived polypeptide) in amino acid sequence, but retains one or morebiological activities described herein of the given polypeptide. Thevariant polypeptide described herein retains at least at least thebiological activities of the polypeptide to bind to NTCP andbidirectionally regulate NTCP-mediated transport of bile acid intohepatocytes. The variant polypeptide described herein may have one ormore amino acid additions (e.g., insertion), deletions, or substitutionsfrom the given polypeptide. In some embodiments, the variant polypeptidedescribed herein may have 1-30, 1-20, 1-10, 1-8, 1-5, or 1-3 amino acidadditions (e.g., insertion), deletions, or substitutions from the givenpolypeptide, including all integers in between these ranges. Forexample, the polypeptide sequence may contain conservative substitutionof amino acids. A conservative substitution of an amino acid, i.e.,replacing an amino acid with a different amino acid of similarproperties (e.g., hydrophilicity and degree and distribution of chargedregions), typically involves a minor change and therefore does notsignificantly alter the biological activity of the polypeptide. Theseminor changes can be identified, in part, by considering the hydropathicindex of amino acids based on a consideration of the hydrophobicity andcharge of the amino acid. Amino acids of similar hydropathic indexes andhydrophilicity values can be substituted and still retain proteinfunction. Both the hydrophobicity index and the hydrophilicity value ofamino acids are influenced by the particular side chain of that aminoacid. Consistent with that observation, amino acid substitutions thatare compatible with biological function depend on the relativesimilarity of the amino acids, and particularly the side chains of thoseamino acids, as revealed by the hydrophobicity, hydrophilicity, charge,size, and other properties.

The term “variant” also includes a polypeptide that has certainidentity, such as, e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the given polypeptide.“Variant” as used herein also includes a polypeptide comprising theportion of the given polypeptide that corresponds to a native sequenceof HBV proteins. “Variant” may also refer to a fusion protein orchimeric protein, comprising polypeptides derived from two or moredifferent sources. Non-limiting examples of the fusion protein describedherein may include, e.g., a fusion protein of one polypeptide derivedfrom HBV and another polypeptide derived from a non-HBV protein, afusion protein of two polypeptides derived from different HBV subtypes,and a fusion protein of two polypeptides derived from different regionsof the L protein of any one of HBV subtypes, or from different sequenceswithin the pre-S1 region of the L protein of any one of HBV subtypes.

The term “variant” also includes a polypeptide that comprises the sameamino acid sequence of a given polypeptide (i.e., the polypeptidedescribed herein, the polypeptide derived from HBV, or the HBV-derivedpolypeptide) and retains one or more biological activities of the givenpolypeptide, but chemically and/or post-translationally modified in amanner different from the given polypeptide. “Variant” can also be usedto describe a polypeptide or a fragment thereof that has beendifferentially processed, such as by proteolysis, phosphorylation, orother post-translational modification, yet retains its biologicalactivity of binding to NTCP and bidirectionally regulating NTCP-mediatedtransport of bile acid into hepatocytes. Use of “variant” herein isintended to encompass fragments of a variant unless otherwisecontradicted by context. The term “variant” also encompasses thehomologous polypeptide sequences found in the different viral species,strains, or subtypes of the hepadnavirus genus. HBV is divided into fourmajor serotypes (adr, adw, ayr, ayw) based on antigenic epitopes presenton its envelope proteins, and into eight genotypes (A-H) according tooverall nucleotide sequence variation of the genome. The term “variant”therefore includes homologous polypeptides found in any of these HBVsubtypes. “Variant” can also include polypeptides having native flankingamino acid sequences from any of these HBV subtypes added to the Nand/or C terminus.

The terms “conservative amino acid substitutions” and “conservativesubstitutions” are used interchangeably herein to refer to intendedamino acid swaps within a group of amino acids wherein an amino acid isexchanged with a different amino acid of similar size, structure,charge, and/or polarity. Families of amino acid residues having similarside chains are known in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, in some embodiments, anamino acid residue in a polypeptide can be replaced with another aminoacid residue from the same side chain family. In other embodiments, astring of amino acids can be replaced with a structurally similar stringthat differs in order and/or composition of side chain family members.In yet other embodiments, mutations can be introduced randomly along allor part of the polypeptide. Examples of conservative amino acidsubstitutions include, e.g., exchange of one of the aliphatic orhydrophobic amino acids Ala, Val, Leu, and Ile for one of the otheramino acids in that group of four; exchange between thehydroxyl-containing residues Ser and Thr; exchange between the acidicresidues Asp and Glu; exchange between the amide residues Asn and Gln;exchange between the basic residues Lys, Arg, and His; exchange betweenthe aromatic residues Phe, Tyr, and Trp; and exchange between thesmall-sized amino acids Ala, Ser, Thr, Met, and Gly. Conservativesubstitutions, such as substituting a conserved amino acid with asimilar, structurally related amino acid would not be reasonablyexpected to impose a substantial influence on the biological activity ofthe polypeptide.

The term “sequence identity” (e.g., a “sequence 50% identical to”)refers to the extent that a sequence is identical on an aminoacid-by-amino acid basis over a window of comparison. In someembodiments, the polypeptide described herein may comprise an amino acidsequence at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of a given polypeptideand still retain one or more biological activities of the givenpolypeptide. A “percentage identity” (or “% identity”) may be calculatedby comparing two optimally aligned sequences over the window ofcomparison, determining the number of positions at which the identicalamino acids occur in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison, and multiplying the result by100 to yield the percentage of sequence identity. Optimal alignment ofsequences for aligning a comparison window may be conducted bycomputerized implementations of algorithms available in the art, suchas, e.g., the BLAST® family of programs, or by visual inspection and thebest alignment (i.e., resulting in the highest percentage homology overthe comparison window) generated by any of the various methods selected.For sequence comparison, one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are input into a computer,subsequence coordinates may be designed, if necessary, and sequencealgorithm program parameters are designated. The sequence comparisonalgorithm then calculates the present sequence identity for the testsequences relative to the reference sequence, based on the designatedprogram parameters. The designation of sequence algorithm programparameters is well within the knowledge in the art. For example, thewindow of comparison may be designated as over the entire length ofeither or both comparison sequences, such as, e.g., over the entirelength of the reference sequence, and gaps of up to 5% of the totalnumber of amino acids in the reference sequence may be allowed.

As used herein, the “biological activity” of the polypeptides describedherein encompasses the ability of the polypeptides to bind to NTCP andbidirectionally regulate NTCP-mediated bile acid uptake in hepatocytes.As used herein, “bidirectional regulation” of a molecule or pathwaymeans that the HBV-derived polypeptides described herein enhances theactivity of the molecule or pathway (i.e., functions as an enhancer) ator below a certain concentration, and inhibits the activity of themolecule or pathway (i.e., functions as an inhibitor) above theconcentration. For instance, the polypeptide described herein may bindto NTCP and promote NTCP-mediated bile acid uptake in hepatocytes (i.e.,functions as an “enhancer” of NTCP) at or below a certain concentration.The same polypeptide may also bind to NTCP but inhibit NTCP-mediatedbile acid uptake (i.e., functions as an “inhibitor” of NTCP) above thatconcentration. In some embodiments, the polypeptide described herein mayfunction as an enhancer of NTCP at or below 93 nmol/L and as aninhibitor of NTCP above 93 nmol/L. For example, the Cmyr-47 polypeptidedescribed herein may function as an enhancer of NTCP at or below 500ng/ml and as an inhibitor of NTCP above 500 ng/ml.

In some embodiments, the polypeptide described herein maybidirectionally regulate NTCP-mediated uptake of bile acids intohepatocytes in vitro. The polypeptide described herein may promote invitro NTCP-mediated bile acid uptake at or below a certainconcentration, while the polypeptide may inhibit in vitro NTCP-mediatedbile acid uptake above that concentration. In some embodiment, thepolypeptide described herein may promote in vitro NTCP-mediated bileacid uptake at or below 93 nmol/L, while the polypeptide may inhibit invitro NTCP-mediated bile acid uptake above 93 nmol/L. For example, theCmyr-47 polypeptide described herein may promote in vitro NTCP-mediatedbile acid uptake at or below 500 ng/ml and inhibit in vitroNTCP-mediated bile acid uptake above 500 ng/ml.

In some embodiments, the polypeptide described herein may promoteNTCP-mediated bile acid uptake in a subject treated with the polypeptideat or below a certain serum concentration of the administeredpolypeptide. The polypeptide may inhibit NTCP-mediated bile acid uptakein a subject treated with the polypeptide above that serum concentrationof the administered polypeptide. In some embodiments, when the serumconcentration of the polypeptide described herein is at or below 93nmol/L in a subject treated with the polypeptide, the polypeptide iscapable of enhancing NTCP-mediated uptake of bile acids in the subject.In some embodiments, when the serum concentration of the polypeptidedescribed herein is above 93 nmol/L in a subject treated with thepolypeptide, the polypeptide is capable of inhibiting NTCP-mediateduptake of bile acids in the subject. For example, the Cmyr-47polypeptide described herein may be capable of enhancing NTCP-mediateduptake of bile acids in the subject at or below a serum concentration of500 ng/ml, and inhibiting NTCP-mediated uptake of bile acids in thesubject above a serum concentration of 500 ng/ml.

The biological activity of the polypeptide described herein may alsoinclude the ability to treat a metabolic disease or to ameliorate one ormore symptoms associated with the metabolic disease. The biologicalactivity may further include the ability of the polypeptide describedherein to prevent the development of a metabolic disease. In someembodiments, the biological activity of the polypeptide described hereinmay include the ability of the polypeptide to modulate the level oractivity of one or more chemical or biological molecules associated withmetabolism, and/or to modulate the level or value of one or morephysiological parameters that measure metabolic changes. In someembodiments, the “biological activity” of the polypeptide describedherein includes the ability of the polypeptide to reduce or stabilizethe level or activity of one or more such chemical or biologicalmolecules or physiological parameters. In some embodiments, metabolismrefers to bile acid metabolism, glucose metabolism, lipid metabolism,and/or amino acid metabolism. The chemical or biological moleculesassociated with metabolism may include, e.g., glucose, triglyceride,cholesterol, free fatty acids, bile acids, amino acids, hormones, suchas, e.g., insulin, LDL-C, HDL-C, HbA1c, blood urea nitrogen, andminerals. The physiological parameters that measure metabolic changesmay include, e.g., glycemia, blood pressure, body weight, fat mass, bodymass index (BMI), inflammation, atherosclerosis index (AI), heart index,kidney index, total fat index, and homeostatic model assessment (HOMA)index.

In certain embodiments, the “biological activity” of the polypeptidedescribed herein includes the ability of the polypeptide to increase thelevel of serum bile acid in a subject. In certain embodiments, the“biological activity” of the polypeptide described herein includes theability of the polypeptide to enhance cholesterol elimination throughbile acid synthesis in hepatocytes.

In some embodiments, the biological activity of the polypeptidedescribed herein may include the ability to lower the serum level of oneor more chemical or biological molecules associated with lipidmetabolism in a subject administered with the polypeptide. In someembodiments, the biological activity of the polypeptides describedherein may include the ability to lower the serum level of serum lipids,such as, e.g., triglyceride, total cholesterol, or LDL-C in a subjectadministered with the polypeptide.

In some embodiments, the biological activity of the polypeptidedescribed herein may include the ability to lower the serum level of oneor more chemical or biological molecules associated with glucosemetabolism in a subject administered with the polypeptide. In someembodiments, the biological activity of the polypeptides describedherein may include the ability to lower the serum level of glucose orHbA1c in a subject administered with the polypeptide. In someembodiments, the polypeptides described herein may be capable ofstabilizing the serum level of insulin in a subject.

In certain embodiments, the “biological activity” of the polypeptidedescribed herein includes the ability of the polypeptide to treat ametabolic disorder in a subject. In some embodiments, the metabolicdisorder involves dysregulation of lipid metabolism. The metabolicdisease may include a cholesterol-related disorder, such as, e.g.,hyperlipidemia (including hypertriglyceridemia, hypercholesterolemia, orboth). In some embodiments, the metabolic disorder involvesdysregulation of glucose metabolism. The metabolic disease may include,e.g., diabetes and obesity.

In certain embodiments, the “biological activity” of the polypeptidedescribed herein includes the ability of the polypeptide to ameliorateor prevent one or more symptoms or complications of such disorders. Incertain embodiments, the “biological activity” of the polypeptidedescribed herein includes the ability of the polypeptide to mitigate thenegative impact of such disorders on the health of a patient or reducethe risk of developing such disorders. In certain embodiments, the“biological activity” of the polypeptide described herein also includesthe ability of the polypeptide to reduce the severity of or the risk ofdeveloping other associated diseases, such as, e.g., atherosclerosisand/or cardiovascular diseases, heart diseases, kidney impairment, orobesity.

Without being bound by theory, it is believed that these biologicalactivities of the polypeptide described herein may result frombidirectional regulation of NTCP-mediated bile acid uptake in thesubject by the polypeptide at the serum concentrations followingadministration of the polypeptide to the subject. In some embodiments,when the concentration of the polypeptide described herein in the bloodstream of a subject administered with such polypeptide is at or below acertain concentration, bile acid uptake in the subject is enhanced. Insome embodiments, when the concentration of the polypeptide describedherein in the blood stream of the subject is above a certainconcentration, bile acid uptake in the subject is inhibited. In someembodiments, when the concentration of the polypeptide described hereinin the blood stream of a subject administered with such polypeptide isat or below 93 nmol/L, bile acid uptake in the subject is enhanced. Insome embodiments, when the concentration of the polypeptide describedherein in the blood stream of the subject is above 93 nmol/L, bile aciduptake in the subject is inhibited. For example, when the concentrationof the Cmyr-47 polypeptide described herein in the blood stream of asubject administered with such polypeptide is at or below 500 ng/ml,bile acid uptake in the subject is enhanced. When the concentration ofthe Cmyr-47 polypeptide described herein in the blood stream of thesubject is above 500 ng/ml, bile acid uptake in the subject isinhibited.

Various in vivo, in vitro, and ex vivo assays to confirm the biologicalactivity of the polypeptide described herein are contemplated. Thebiological activity of the polypeptide described herein may be confirmedin vivo, by collecting a sample from a subject treated with thepolypeptide described herein. The sample may be a biopsy samplecollected from a specific tissue such as, e.g., liver, muscle, fat, andpancreas, or a snap-frozen tissue collected from an animal post-mortem.In some embodiments, the sample may be a serum sample collected fromblood drawn from a subject. Various methods for collecting a serumsample from a subject are known in the art, and include, e.g.,tail-bleeding, retro-orbital puncture, and cardiopuncture. In someembodiments, the biological activity of the polypeptide described hereinmay be confirmed in vitro, by contacting the polypeptide describedherein with a cell that is either a transformed cell line or a cellisolated from an animal. In some embodiments, the cell may be a primaryhepatocyte isolated from an animal.

Various methods can be used to confirm the ability of the polypeptidedescribed herein to bidirectionally regulate NTCP in a quantitativemanner. For instance, cells expressing NTCP (e.g., mammalian cellsoverexpressing NTCP or hepatocytes) may be treated in vitro with bileacids and increasing amounts of the polypeptide described herein. Bileacids added to the cells may be radiolabeled or chemically labeled fordetection. The cells may be then harvested and the amount of bile acidstaken up by the cells may be measured. The ability of the polypeptidedescribed herein to bidirectionally regulate NTCP may be confirmed whenthe polypeptide enhances bile acid uptake at or below a certainconcentration while inhibits bile acid uptake above that concentration.

The exemplary assays useful to confirm the biological activity of thepolypeptide may also include a functional analysis with a samplecollected from a subject treated with the polypeptide described herein,including, e.g., glucose production assay, glucose uptake assay, fattyacid oxidation assay, cholesterol assay, bile acids assay, urea assay,and triglyceride assay. In some embodiments, the assays may alsoinclude, e.g., a binding analysis between the polypeptide and NTCP, anactivity assay of NTCP for transporting bile acids, and an expression,localization, or activity analysis of molecular factors involved inmetabolism, such as, e.g., bile acid metabolism, glucose metabolism,lipid metabolism, and amino acid metabolism. The foregoing techniquesand procedures to confirm the biological activity of the polypeptidesdescribed herein may be performed by following methods known in the artand procedures provided in this specification.

In some embodiments, the polypeptide described herein may comprise anamino acid sequence of the pre-S1 region of any HBV subtype. In someembodiments, the polypeptide described herein comprises the sequence ofamino acids 13-59 of the pre-S1 region of HBV genotype C:GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG (SEQ ID NO: 23).

In additional embodiments, the polypeptide described herein may comprisethe corresponding pre-S1 sequence from another HBV genotype, such as,e.g., any one of genotypes A, B, D, E, F, G, and H. For example, in someembodiments, the polypeptide described herein may comprise:

pre-S1 amino acids 13-59 of HBV genotype A: (SEQ ID NO: 34)GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPVKDDWPAANQVG,pre-S1 amino acids 13-59 of HBV genotype B: (SEQ ID NO: 35)GTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPNKDNWPDANKVG,pre-S1 amino acids 2-48 of the HBV genotype D: (SEQ ID NO: 36)GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVG,pre-S1 amino acids 12-58 of the HBV genotype E: (SEQ ID NO: 37)GKNISTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDHWTEANKVG,pre-S1 amino acids 13-59 of the HBV genotype F: (SEQ ID NO: 38)GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDSWPMANKVG,pre-S1 amino acids 12-58 of the HBV genotype G: (SEQ ID NO: 39)GKNLSASNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVG, orpre-S1 amino acids 13-59 of the HBV genotype H: (SEQ ID NO: 40)GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDNWPMANKVG.

In some embodiments, the polypeptide described herein may comprise aportion of the pre-S1 region of HBV, said portion comprising at least anamino acid sequence chosen from SEQ ID NOs: 23 and 34-40. In someembodiments, the polypeptide described herein may comprise the entirepre-S1 region of HBV.

In some embodiments, the polypeptide described herein may be 10-100amino acids in length. For example, the polypeptide may be 15-100,15-80, 20-100, 20-80, 20-60, 25-60, 30-60, 35-60, or 40-60 amino acidsin length, including all integers in between these ranges. In someembodiments, the polypeptide described herein may be at least 20, suchas, e.g., at least 25, 30, 35, 40, amino acids in length. In someembodiments, the polypeptide described herein may be 20, 25, 30, 35, 40,47, 55, 60 amino acids in length. In some embodiments, the polypeptidedescribed herein may be 47 amino acids in length. The variants of thepolypeptides described herein that differ in length retain one or morebiological activities associated with the corresponding polypeptides,including at least the biological activity of binding to NTCP andbidirectionally regulating NTCP-mediated transport of bile acid intohepatocytes.

In some embodiments, the polypeptide described herein may comprise anN-terminal modification with a hydrophobic group. For example, thehydrophobic group may be chosen from myristic acid, palmitic acid,stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonicacid. In some embodiments, the hydrophobic group may be chosen frommyristic acid, palmitic acid, stearic acid, and cholesterol. In someembodiments, the hydrophobic group may be myristic acid. In certainembodiments, the polypeptide described herein may comprise an amino acidsequence chosen from SEQ ID NOs: 23 and 34-40, wherein the N terminusmay be modified with a hydrophobic group chosen from myristic acid,palmitic acid, stearic acid, and cholesterol. In certain embodiments,the polypeptide described herein may comprise an amino acid sequencechosen from SEQ ID NOs: 23 and 34-40, wherein the N terminus may bemyristoylated. In some embodiments, the polypeptide described herein maycomprise the amino acid sequence of SEQ ID NO: 23, wherein the Nterminus may be myristoylated. In some embodiments, the polypeptidedescribed herein may comprise a C-terminal modification to stabilize thepolypeptide. For example, the C-terminal modification may be chosen fromamidation (amination), isopentanediolization, and any other C-terminalmodification capable of stabilizing the polypeptide described herein. Insome embodiments, the C-terminal modification may be amidation(amination). For example, the polypeptide described herein may comprisethe amino acid sequence of NO: 23, wherein the N terminus may bemyristoylated, and/or the C terminus may be amidated (aminated). In someembodiments, the polypeptide described herein may comprise the aminoacid sequence of NO: 3 (Cmyr-47). In some embodiments, the polypeptidedescribed herein may comprise an amino acid sequence chosen from SEQ IDNOs: 34-40, wherein the N terminus may be myristoylated, and/or the Cterminus may be modified by amidated (aminated). In some embodiments,the polypeptide described herein may comprise an amino acid sequencechosen from SEQ ID NOs: 14-20. The variants of the polypeptide describedherein that are modified at the N-terminus and/or the C-terminus retainone or more biological activities of the corresponding polypeptides thatare not modified in the same manner, including at least the biologicalactivity of binding to NTCP and bidirectionally regulating NTCP-mediatedtransport of bile acid into hepatocytes.

Variants of the polypeptides described herein are also contemplated inthe present disclosure, including variants with one or more amino aciddeletions, substitutions, or insertions that retain one or morebiological activities of the polypeptides, including at least thebiological activity of binding to NTCP and bidirectionally regulatingNTCP-mediated transport of bile acid into hepatocytes. The polypeptidesdescribed herein preferably retain the glycine corresponding to aminoacid 13 of the pre-S1 region of HBV genotype C (i.e., the N-terminalglycine of SEQ ID NO: 23). In some embodiments, the polypeptidesdescribed herein retain the asparagine corresponding to amino acid 20 ofthe pre-S1 region of HBV genotype C. In some embodiments, thepolypeptide described herein may have one or more naturally-occurringmutations in the pre-S1 region of HBV. In some embodiments, thepolypeptide described herein may have 1-30, such as, e.g., 1-20, 1-10,1-8, 1-5, or 1-3, amino acid deletions, substitutions, or insertionsrelative to a sequence from the pre-S1 region of HBV, including allintegers in between these ranges. In some embodiments, the polypeptidedescribed herein may have 1-30, such as, e.g., 1-20, 1-10, 1-8, 1-5, or1-3, amino acid deletions, substitutions, or insertions relative to anamino acid sequence chosen from SEQ ID NOs: 23 and 34-40, including allintegers in between these ranges. In some embodiments, the polypeptidedescribed herein may have 1-30, such as, e.g., 1-20, 1-10, 1-8, 1-5, or1-3, amino acid deletions, substitutions, or insertions relative to theamino acid sequence of SEQ ID NO: 23, including all integers in betweenthese ranges. In some embodiments, the polypeptide described herein mayhave 1-3 amino acid deletions, substitutions, or insertions from theamino acid sequence of SEQ ID NO: 23. In certain embodiments, thepolypeptide described herein may have 1-30, such as, e.g., 1-20, 1-10,1-8, 1-5, or 1-3, amino acid deletions or insertions at the C terminusof an amino acid sequence chosen from SEQ ID NOs: 23 and 34-40,including all integers in between these ranges. For example, thepolypeptide described herein may comprise an amino acid sequence chosenfrom SEQ ID NOs: 21, 22, and 24-28. In some embodiments, the polypeptidedescribed herein may comprise the amino acid sequence of any one of thepolypeptides listed in Table 1. In some embodiments, the polypeptidedescribed herein may be chosen from any one of the post-translationallymodified polypeptides listed in Table 1.

TABLE 1 List of Exemplary Polypeptides SEQ N-terminal C-terminal IDModifi- Amino acid sequence Modifi- No. SEQ name cation123456789012345678901234567890 cation SEQ origin 1 Cmyr-60 MyrGTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype CWDFNPNKDHWPFANQVGAGAFGPGFTPPHG Pre-S1(13-72) 2 Cmyr-55 MyrGTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype C WDFNPNKDHWPEANQVGAGAFGPGFPre-S1(13-67) 3 Cmyr-47 Myr GTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2Genotype C WDFNPNKDHWPEANQVG Pre-S1(13-59) 4 Cmyr-40 MyrGTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype C WDFNPNKDHW Pre-S1(13-52) 5Cmyr-35 Myr GTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype C WDFNPPre-S1(13-47) 6 Cmyr-30 Myr GTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2Genotype C Pre-S1(13-42) 7 Cmyr-25 Myr GTNSVPNPLGFFPDHQLDPAFGAN NH2Genotype C Pre-S1(13-37) 8 Cmyr-20 Myr GTNSVPNPLGETPDHQLDP NH2Genotype C Pre-S1(13-32) 9 Cmyr-47+ Myr GGWSSKPRQGMGTNLSVPNPLGFFPDHQLDNH2 Genotype C (−10)  PAFGANSNNPDWDFNPNKDHWPEANQVG Pre-S1(2-59) 10Cmyr-47+ Myr GLSWTVPLEGTNLSVPNPLGFFPDHQLDP NH2 Genotype E or (−9)AFGANSNNPDWDFNPNKDHWPEANQVG G Pre-S1(2-11) + Genotype C Pre-S1(13-59) 11Cplam-47 Plam GTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype CWDFNPNKDHWPEANQVG Pre-S1(13-59) 12 Cstea-47 StearoylGTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype C WDFNPNKDHWPEANQVGPre-S1(13-59) 13 Cchol-47 Chol GTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2Genotype C WDFNPNKDHWPEANQVG Pre-S1(13-59) 14 Amyr-47 MyrGTNLSVPNPLGFFPDHQLDPAFGANSNNPD NH2 Genotype A WDFNPVKDDWPAANQVGPre-S1(13-59) 15 Bmyr-47 Myr GTNLSVPNPLGFFPDHQLDPAFKANSENPD NH2Genotype B WDLNPNKDNWPDANKVG Pre-S1(13-59) 16 Dmyr-47 MyrGQNLSTSNPLGFFPDHQLDPAFRANTANPD NH2 Genotype D WDFNPNKDTWPDANKVGPre-S1(2-48) 17 Emyr-47 Myr GKNISTTNPLGFFPDHQLDPAFRANTRNPD NH2Genotype E WDHNPNKDHWTEANKVG Pre-S1(12-58) 18 Fmyr-47 MyrGQNLSVPNPLGFFPDHQLDPLFPANSSSPD NH2 Genotype F WDFNTNKDSWPKANKVGPre-S1(13-59) 19 Gmyr-47 Myr GKNLSASNPLGFLPDHQLDPAFRANTNNPD NH2Genotype G WDFNPKKDPWPEANKVG Pre-S1(12-58) 20 Hmyr-47 MyrGQNLSVPNPLGFFPDHQLDPLFRANSSSPD NH2 Genotype H WDFNTNKDNWPMANKVGPre-S1(13-59)

In various embodiments, the polypeptide described herein may have atleast about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% identity to any of the polypeptides described herein. Forexample, the polypeptide may comprise an amino acid sequence at leastabout 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 21-40. In some embodiments, thepolypeptide may comprise an amino acid sequence at least about 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toany one of SEQ ID NOs: 23 and 34-40. In some embodiments, thepolypeptide may comprise an amino acid sequence at least about 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 23. The variants having certain sequence identity to thepolypeptides described herein retain one or more biological activitiesof the corresponding polypeptides, including at least the biologicalactivity of binding to NTCP and bidirectionally regulating NTCP-mediatedtransport of bile acid into hepatocytes.

Aspects of the present disclosure also include variants of thepolypeptides described herein having a native flanking amino acidsequence from the HBV L protein, such as, e.g., from the pre-S1 regionof the L protein, added to the N and/or C terminus. The native flankingamino acid sequence refers to the native sequence flanking the N or Cterminus of the polypeptide described herein in the pre-S1 region of thecorresponding HBV genotype or any other HBV genotypes. In someembodiments, the polypeptide described herein may comprise an amino acidsequence chosen from SEQ ID NOs: 23 and 34-40, and a native flankingamino acid sequence at the N and/or C terminus derived from the pre-S1region of any one of HBV genotypes A-H. In some embodiments, the nativeflanking amino acid sequence may be derived from the consensus sequenceof an HBV strain with the GenBank Accession No. KC875260 (genotype A;SEQ ID NO: 41), AY220704 (genotype B; SEQ ID NO: 42), AF461363 (genotypeC; SEQ ID NO: 43), AY796030 (genotype D; SEQ ID NO: 44), AB205129(genotype E; SEQ ID NO: 45), DQ823095 (genotype F; SEQ ID NO: 46),HE981176 (genotype G; SEQ ID NO: 47), or AB179747 (genotype H; SEQ IDNO: 48). For example, the polypeptide described herein may comprise theamino acid sequence of SEQ ID NO: 23, and a native flanking amino acidsequence at the N and/or C terminus derived from the pre-S1 region ofHBV genotype C. Alternatively, the polypeptide described herein maycomprise the amino acid sequence of SEQ ID NO: 23, and a native flankingamino acid sequence at the N and/or C terminus derived from the pre-S1region of any one of HBV genotypes A, B, D, E, F, G, and H. In someembodiments, the N and/or C terminus of the polypeptide described hereinmay independently comprise a native flanking amino acid sequence havinga length of 1-10, such as, e.g., 1-8, 1-5, or 1-3 amino acids, includingall integers in between these ranges. For example, the polypeptidedescribed herein may comprise the amino acid sequence of SEQ ID NO: 23and a native flanking amino acid sequence of 10 amino acids at the Nterminus from the pre-S1 region of HBV genotype C. In other words, thepolypeptide may comprise amino acids 2-59 of the pre-S1 region of HBVgenotype C (SEQ ID NO: 29). As another example, the polypeptidedescribed herein may comprise the amino acid sequence of SEQ ID NO: 23and a native flanking amino acid sequence of 9 amino acids at the Nterminus from the pre-S1 region of HBV genotype E or G. In other words,the polypeptide may comprise amino acids 13-59 of the pre-S1 region ofHBV genotype C and amino acids 2-11 of the pre-S1 region of HBV genotypeE or G (SEQ ID NO: 30). It will be appreciated that, any polypeptidesdescribed herein can have native flanking amino acid sequences of anylength extended from the N and/or C terminus, and the resultingpolypeptides retain one or more biological activities of the originalpolypeptides, including at least the biological activity of binding toNTCP and bidirectionally regulating NTCP-mediated transport of bile acidinto hepatocytes.

In some embodiments, the polypeptides described herein are capable ofbidirectionally regulating NTCP-mediated transport of bile acids intohepatocytes. When hepatocytes are in contact with the polypeptidedescribed herein at or below a certain concentration, NTCP-mediatedtransport of bile acid into the hepatocytes may be enhanced as comparedwith hepatocytes that are not in contact with such polypeptide. Whenhepatocytes are in contact with the polypeptide described herein abovethe certain concentration, NTCP-mediated transport of bile acid into thehepatocytes may be inhibited as compared with hepatocytes that are notin contact with such polypeptide. In some embodiments, when hepatocytesare in contact with at or below 93 nmol/L of the polypeptide describedherein, NTCP-mediated transport of bile acid into the hepatocytes may beenhanced as compared with hepatocytes that are not in contact with suchpolypeptide. In some embodiments, when hepatocytes are in contact withabove 93 nmol/L of the polypeptide described herein, NTCP-mediatedtransport of bile acid into the hepatocytes may be inhibited as comparedwith hepatocytes that are not in contact with such polypeptide. Forexample, when hepatocytes are in contact with at or below 500 ng/ml ofthe Cmyr-47 polypeptide described herein, NTCP-mediated transport ofbile acid into the hepatocytes may be enhanced as compared withhepatocytes that are not in contact with such polypeptide. In someembodiments, when hepatocytes are in contact with above 500 ng/ml of theCmyr-47 polypeptide described herein, NTCP-mediated transport of bileacid into the hepatocytes may be inhibited as compared with hepatocytesthat are not in contact with such polypeptide.

In some embodiments, the polypeptides described herein may be capable ofmodulating, such as, e.g., reducing or stabilizing, the level oractivity of one or more chemical or biological molecules associated withmetabolism in a subject. In some embodiments, metabolism may include,e.g., bile acid metabolism, glucose metabolism, lipid metabolism, oramino acid metabolism. In some embodiments, the chemical or biologicalmolecule is chosen from glucose, triglyceride, cholesterol, free fattyacids, bile acids, amino acids, hormones, LDL-C, HDL-C, HbA1c, bloodurea nitrogen, and minerals. In some embodiments, the polypeptidesdescribed herein may be capable of modulating, such as, e.g., reducingor stabilizing, the level or value of one or more physiologicalparameters that measure metabolic changes such as, e.g., glycemia, bloodpressure, body weight, fat mass, body mass index (BMI), inflammation,atherosclerosis index (AI), heart index, kidney index, total fat index,and homeostatic model assessment (HOMA) index. In some embodiments, thepolypeptides described herein may be capable of increasing the serumlevel of bile acids in a subject. In some embodiments, the polypeptidesdescribed herein may be capable of reducing the level of serum lipids ina subject. In some embodiments, the polypeptides described herein may becapable of reducing the serum level of total cholesterol in a subject.In further embodiments, the polypeptides described herein may be capableof reducing the serum level of LDL-cholesterol in a subject. In someembodiments, the polypeptides described herein may be capable ofreducing the serum level of triglyceride in a subject. In someembodiments, the polypeptides described herein may be capable ofreducing the serum level of glucose in a subject. In some embodiments,the polypeptides described herein may be capable of reducing the serumlevel of HbA1c in a subject. In some embodiments, the polypeptidesdescribed herein may be capable of stabilizing the serum level ofinsulin in a subject. In some embodiments, the subject may be a mammal.In some embodiments, the subject may be a human. In some embodiments,the subject may suffer from a metabolic disease or may be at risk ofdeveloping such disease.

As used herein, “modulate” or “alter,” all used interchangeably,includes “reducing,” “decreasing,” “lowering,” “down-regulating,” or“inhibiting” one or more quantifiable parameters optionally by a definedand/or statistically significant amount. The term “modulate” alsoincludes “enhancing,” “increasing,” “elevating,” “up-regulating,” or“promoting” one or more quantifiable parameters optionally by a definedand/or statistically significant amount.

The terms “reduce,” “decrease,” “lower,” “down-regulate,” and “inhibit,”all used interchangeably herein, mean that the level or activity of oneor more chemical or biological molecules associated with metabolism suchas, e.g., glucose, triglyceride, cholesterol, free fatty acids, bileacids, amino acids, or hormones, including, e.g., insulin, LDL-C, HDL-C,HbA1c, blood urea nitrogen, and minerals, is reduced below the level oractivity observed in the absence of the polypeptides described herein orlower than a control polypeptide. In some embodiments, “reduce” may meanthat the level or value of one or more physiological parameters thatmeasure metabolic changes, such as, e.g., glycemia, blood pressure, bodyweight, fat mass, body mass index (BMI), inflammation, atherosclerosisindex (AI), heart index, kidney index, total fat index, and homeostaticmodel assessment (HOMA) index, are reduced below the level or activityobserved in the absence of the polypeptides described herein or lowerthan a control polypeptide. In certain embodiments, reduction with apolypeptide described herein is below the level or activity observed inthe presence of an inactive or attenuated molecule. In some embodiments,the polypeptides described herein are capable of reducing the level ofglucose, insulin, cholesterol, or triglyceride in the serum and/or inanother tissue or organ, such as, e.g., liver, heart, muscle, visceralfat, subcutaneous fat, intestine, and brain.

As used herein, the value of body mass index (BMI) of a subject can becalculated with the following formula: BMI=(Mass of the subjectexpressed in kg)/[(height of the subject expressed in m)²]. The level ofinflammation can be measured by following various clinical testsavailable in the art. For instance, the level of C-reactive protein(CRP) in blood can be measured to quantitatively measure the level ofinflammation in a subject. An erythrocyte sedimentation rate (ESR) testis another example of tests that measure the level of inflammation in asubject. The ESR test measures the rate of erythrocytes sediment in aset period. Upon obtaining the levels (such as, e.g., in mmol/L) oftotal cholesterol (TC) and HDL-C in a subject, the value ofatherosclerosis index (AI) can be calculated by following the formula ofAI=(TC−HDL-C)/HDL-C. As used herein, homeostatic model assessment (HOMA)index may refer HOMA-IR (quantifying the level of insulin resistance)index and/or HOMA-β index (quantifying the level of β-cell function).The value of HOMA-IR can be calculated by following the formula of:HOMA-IR=[(blood glucose expressed in mmol/L)×(serum insulin expressed inmU/L)]/22.5. The value of HOMA-β can be calculated by following theformula of: HOMA-β=[(20×serum insulin expressed in mU/L)/(blood glucoseexpressed in mmol/L−3.5)]%. The value of heart index refers to the ratiobetween the weight of heart and the total body weight and can becalculated by following the formula of: heart index (g/kg)=weight ofheart in g/body weight in kg. The value of kidney index refers to theratio between the weight of kidney and the total body weight and can becalculated by following the formula of: kidney index (g/kg)=weight ofkidney in g/body weight in kg. The value of total fat index refers tothe ratio between the weight of fat (e.g., abdominal and/or scapularfat) and the total body weight. As used herein, the fat index can becalculated by following the formula of: total fat index (g/kg)=totalweight of abdominal fat and scapular fat in g/body weight in kg.

Likewise, the terms “enhance,” “increase,” “elevate,” “up-regulate,” and“promote” all used interchangeably, mean that the level or activity ofone or more chemical or biological molecules associated with metabolism,such as, e.g., glucose, triglyceride, cholesterol, free fatty acids,bile acids, amino acids, hormones, including, e.g., insulin, LDL-C,HDL-C, HbA1c, blood urea nitrogen, and minerals, is increased above thelevel or activity observed in the absence of the polypeptides describedherein or higher than a control polypeptide. In some embodiments,“enhance” may mean that the level of value of one or more physiologicalparameters that measure metabolic changes, such as, e.g., glycemia,blood pressure, body weight, fat mass, body mass index (BMI),inflammation, atherosclerosis index (AI), heart index, kidney index,total fat index, and homeostatic model assessment (HOMA) index, areincreased above the level or activity observed in the absence of thepolypeptides described herein or higher than a control polypeptide. Incertain embodiments, increase with a polypeptide described herein isabove the level or activity observed in the presence of an inactive orattenuated molecule. In some embodiments, the polypeptides describedherein are capable of increasing the level of bile acid in the serumand/or in another tissue or organ, such as, e.g., liver, heart, muscle,visceral fat, subcutaneous fat, intestine, and brain.

The terms “stabilize,” “maintain,” “sustain,” and “preserve,” are usedinterchangeably in connection with one or more chemical or biologicalmolecules associated with metabolism, and mean that the level oractivity of the one or more chemical or biological molecules associatedwith metabolism, such as, e.g., glucose, triglyceride, cholesterol, freefatty acids, bile acids, amino acids, hormones, including, e.g.,insulin, LDL-C, HDL-C, HbA1c, blood urea nitrogen, and minerals, shows aminimal difference from the level or activity observed in a healthysubject or a subject who is not suffering from a metabolic disease, orfrom the level or activity observed in the presence of a positivecontrol polypeptide. In some embodiments, “stabilize” may mean that thelevel or value of one or more physiological parameters that measuremetabolic changes, such as, e.g., glycemia, blood pressure, body weight,fat mass, body mass index (BMI), inflammation, atherosclerosis index(AI), heart index, kidney index, total fat index, and homeostatic modelassessment (HOMA) index, shows a minimal difference from the level orvalue observed in a healthy subject or a subject who is not sufferingfrom a metabolic disease, or from the level or value observed in thepresence of a positive control polypeptide. In some embodiments, thepolypeptides described herein are capable of stabilizing the level ofinsulin in the serum and/or insulin production from pancreas.

The polypeptides described herein can be made by chemical synthesis orby employing recombinant technology.

When recombinant procedures are selected, a synthetic gene may beconstructed de novo or a natural gene may be mutated by, for example,cassette mutagenesis. The polypeptides described herein may be producedusing recombinant DNA techniques. These techniques contemplate, insimplified form, taking the gene, either natural or synthetic, encodingthe peptide; inserting it into an appropriate vector; inserting thevector into an appropriate host cell; culturing the host cell to causeexpression of the gene; and recovering or isolating the peptide producedthereby. In some embodiments, the recovered peptide is then purified toa suitable degree.

For example, the DNA sequence encoding a polypeptide described herein iscloned and manipulated so that it may be expressed in a convenient host.DNA encoding parent polypeptides can be obtained from an HBV genomiclibrary, from cDNA derived from mRNA from cells expressing thepolypeptide, or by synthetically constructing the DNA sequence. Theparent DNA is then inserted into an appropriate plasmid or vector whichis used to transform a host cell. In general, plasmid vectors containingreplication and control sequences which are derived from speciescompatible with the host cell are used in connection with those hosts.The vector ordinarily carries a replication site, as well as sequenceswhich encode proteins or peptides that are capable of providingphenotypic selection in transformed cells. The vector may be thosecommonly used in the art, or constructed using standard techniques bycombining functional fragments of the vectors commonly used in the art.

The host cell may be prokaryotic or eukaryotic. For example, prokaryotichost cells may include E. coli, Bacillus subtilis, and otherenterobacteriaceae such as, e.g., Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species. In addition to prokaryotes,eukaryotic organisms, such as yeast cultures, or cells derived frommulticellular organisms, such as insect or mammalian cell cultures, maybe used. Examples of such eukaryotic host cell lines include VERO andHeLa cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK,COS-7, and MDCK cell lines.

In some embodiments, the polypeptides described herein may be preparedusing solid-phase synthesis, or other equivalent chemical synthesesknown in the art. In some embodiments, solid-phase synthesis isinitiated from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a BHA resin or IBHA resin. The amino acids are coupled tothe peptide chain using techniques well known in the art for theformation of peptide bonds. One method involves converting the aminoacid to a derivative that will render the carboxyl group moresusceptible to reaction with the free N-terminal amino group of thepeptide fragment. For example, the amino acid can be converted to amixed anhydride by reaction of a protected amino acid withethylchloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, pivaloyl chloride or like acid chlorides.Alternatively, the amino acid can be converted to an active ester suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole. Another couplingmethod involves use of a suitable coupling agent such asN,N′-dicyclohexylcarbodiimide or N,N′-diisopropylcarbodiimide.

In some embodiments, the α-amino group of each amino acid employed inthe peptide synthesis may be protected during the coupling reaction toprevent side reactions involving their active α-amino function. Forexample, certain amino acids that contain reactive side-chain functionalgroups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl) may beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at that site during both the initial and subsequentcoupling steps. The selection of a suitable side-chain protecting groupis within the skill of the art. The protecting group will be readilyremovable upon completion of the desired amino acid peptide underreaction conditions that will not alter the structure of the peptidechain.

After removal of the α-amino protecting group, the remaining α-amino andside-chain protected amino acids are coupled stepwise within the desiredorder. As an alternative to adding each amino acid separately in thesynthesis, some may be coupled to one another prior to addition to thesolid-phase synthesizer. The selection of an appropriate couplingreagent is within the skill of the art.

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in excess, and the coupling is suitably carried outin a medium of dimethylformamide (DMF) or CH₂Cl₂ or mixtures thereof. Ifincomplete coupling occurs, the coupling procedure is repeated beforeremoval of the N-amino protecting group prior to the coupling of thenext amino acid. The success of the coupling reaction at each stage ofthe synthesis may be monitored. The coupling reactions can be performedautomatically using well known methods, for example, a BIOSEARCH 9500™peptide synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting groupsis suitably accomplished simultaneously or stepwise. When the resinsupport is a chloro-methylated polystyrene resin, the bond anchoring thepeptide to the resin is an ester linkage formed between the freecarboxyl group of the C-terminal residue and one of the manychloromethyl groups present on the resin matrix. It will be appreciatedthat the anchoring bond can be cleaved by reagents that are known to becapable of breaking an ester linkage and of penetrating the resinmatrix. It will also be recognized that the polypeptides may be modified(such as, e.g., modified at the N-terminus with a hydrophobic group,including, e.g., myristic acid, palmitic acid, stearic acid, oleic acid,linoleic acid, cholesterol, arachidonic acid; modified at the C-terminusby amidation (amination), isopentanediolization, or other stabilizingC-terminal modification) either before or after the polypeptide iscleaved from the support.

Purification of the polypeptides of the invention may be achieved usingconventional procedures such as preparative HPLC (including reversedphase HPLC) or other known chromatographic techniques such as gelpermeation, ion exchange, partition chromatography, affinitychromatography (including monoclonal antibody columns) or countercurrentdistribution.

II. Pharmaceutical Compositions

The present disclosure also provides compositions, includingpharmaceutical compositions, comprising a polypeptide described herein.In certain embodiments, the composition may comprise any one or morepolypeptides described herein. In some embodiments, the composition mayfurther comprise a suitable pharmaceutically acceptable carrier. In someembodiments, when administered to a subject in need thereof, thepharmaceutical composition provides serum concentrations of thepolypeptide described herein that allow for bidirectional regulation ofNTCP-mediated bile acid uptake in the subject.

A “pharmaceutically acceptable carrier” refers to an inactiveingredient, such as, e.g., solid, semisolid, or liquid filler, diluent,encapsulating material, formulation auxiliary, excipient, or carrier,for use with a therapeutic agent that together comprise a“pharmaceutical composition” for administration to a subject. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed, and is compatible with otheringredients of the formulation. The pharmaceutically acceptable carrieris appropriate for the formulation employed. For example, if thetherapeutic agent is to be administered orally, the carrier may be a gelcapsule. If the therapeutic agent is to be administered subcutaneously,the carrier ideally is not irritable to the skin and does not causeinjection site reaction.

Pharmaceutical compositions of the polypeptides described herein may beprepared by mixing such polypeptide having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers may include, e.g.: buffers (suchas, e.g., phosphate, citrate, and other organic acids); antioxidants(such as, e.g., ascorbic acid and methionine); preservatives (such as,e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride, benzethonium chloride, phenol, butyl or benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecularweight (such as, e.g., less than about 10 residues) polypeptides;proteins (such as, e.g., serum albumin, gelatin, or immunoglobulins);hydrophilic polymers (such as, e.g., polyvinylpyrrolidone); amino acids(such as, e.g., glycine, glutamine, asparagine, histidine, arginine, orlysine); monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents (such as,e.g., EDTA); sugars (such as, e.g., sucrose, mannitol, trehalose orsorbitol); salt-forming counter-ions (such as, e.g., sodium); metalcomplexes (such as, e.g., Zn-protein complexes); and/or non-ionicsurfactants (such as, e.g., polyethylene glycol (PEG)).

Exemplary pharmaceutical carriers may also include binding agents (suchas, e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose, etc.); fillers (such as, e.g., lactoseand other sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (such as, e.g., magnesium stearate, talc, silica,colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (such as, e.g., starch,sodium starch glycolate, etc.); and wetting agents (such as, e.g.,sodium lauryl sulphate, etc.).

Exemplary pharmaceutically acceptable carriers may further includeinterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). In some embodiments, a sHASEGP may be combined inthe pharmaceutical composition with one or more additionalglycosammoglycanases, such as, e.g., chondroitinases.

The pharmaceutical compositions may also comprise more than one activeingredient suitable for the particular indication being treated, forexample, those with complementary activities that do not adverselyaffect each other. Such active ingredients may be suitably present incombination in amounts that are effective for the purpose intended.

In some embodiments, the active ingredients may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, such as, e.g., hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (such as e.g.,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions.

In some embodiments, the pharmaceutical composition may comprisesustained-release preparations. Suitable examples of sustained-releasepreparations include, e.g., semipermeable matrices of solid hydrophobicpolymers containing the polypeptides described herein, which matricesmay be in the form of shaped articles, such as, e.g., films ormicrocapsules.

In some embodiments, the pharmaceutical compositions may be used for invivo administration and may be sterile. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.

The pharmaceutical compositions may be formulated into any of manypossible dosage forms, such as, e.g., tablets, capsules, gel capsules,powders, or granules. The pharmaceutical compositions may also beformulated as solutions, suspensions, emulsions, or mixed media. In someembodiments, the pharmaceutical compositions may be formulated aslyophilized formulations or aqueous solutions.

In some embodiments, the pharmaceutical compositions may be formulatedas a solution. For example, the polypeptides described herein may beadministered in an unbuffered solution, such as, e.g., in saline or inwater. In some embodiments, the polypeptides may also be administered ina suitable buffer solution. For example, the buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In some embodiments, the buffer solution may bephosphate buffered saline (PBS). The pH and osmolality of the buffersolution containing the polypeptides can be adjusted to be suitable foradministering to a subject.

In some embodiments, the pharmaceutical compositions may be formulatedas suspensions in aqueous, non-aqueous, or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions may be formulatedas emulsions. Exemplary emulsions include heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 m in diameter. Emulsions may contain additional components inaddition to the dispersed phases, and the active drug which may bepresent in a solution in the aqueous phase, the oily phase, or itself asa separate phase. Microemulsions are also included as an embodiment ofthe present disclosure. In some embodiments, the pharmaceuticalcompositions may also be formulated as liposomal formulations.

III. Methods of Use

Embodiments of the present disclosure include therapeutic uses of thepolypeptides described herein. In one aspect, use of the polypeptidesdescribed herein as a medicament is provided. In another aspect, use ofthe polypeptides described herein in treating a metabolic disease isprovided. In some embodiments, the metabolic disease involvesdysregulation of lipid metabolism. In certain embodiments, the metabolicdisease may be a cholesterol-related disorder. In some embodiments, thecholesterol-related disorder may be hyperlipidemia. In some embodiments,the hyperlipidemia may be hypertriglyceridemia, hypercholesterolemia, ora combination thereof. In some embodiments, use of the polypeptidesdescribed herein in treating conditions associated with elevated serumlevel of any one of total triglycerides, total cholesterol, and LDL-C isprovided.

In some embodiments, the metabolic disease involves dysregulation ofglucose metabolism. In some embodiments, the metabolic disease isdiabetes. In some embodiments, the metabolic disease is type IIdiabetes. In some embodiments, the metabolic disease is obesity. In someembodiments, use of the polypeptides described herein in treatingconditions associated with elevated serum level of glucose or HbA1c isalso provided.

In another aspect, a method of treating a metabolic disease in a subjectis provided, comprising administering to the subject a therapeuticallyeffective amount of the polypeptides described herein or of apharmaceutical composition of such polypeptide. In certain embodiments,the subject may suffer from a metabolic disease or may be at risk ofdeveloping such disease. In some embodiments, the subject may be amammal. In some embodiments, the subject may be a human. In certainembodiments, the methods and uses described herein may further compriseadministering to the subject a therapeutically effective amount of atleast one additional therapeutic agent.

In some embodiments, the metabolic disease involves dysregulation oflipid metabolism. In some embodiments, the metabolic disease may be acholesterol-related disorder. In some embodiments, thecholesterol-related disorder may be hyperlipidemia. In some embodiments,the hyperlipidemia may be hypertriglyceridemia, hypercholesterolemia, ora combination thereof.

In other aspects, a method of lowering the level of serum lipids, suchas, e.g., the total cholesterol, total triglyceride, or LDL cholesterollevel, in a subject in need thereof is provided, comprisingadministering to the subject a therapeutically effective amount of thepolypeptide described herein or of a pharmaceutical composition of suchpolypeptide. In some embodiments, the subject is suffering or at risk ofdeveloping a metabolic disease involving dysregulation of lipidmetabolism. In some embodiments, the subject is suffering or at risk ofdeveloping a cholesterol-related disorder. In some embodiments, thesubject is suffering or at risk of developing hyperlipidemia. In someembodiments, the subject is suffering or at risk of developinghypertriglyceridemia, hypercholesterolemia, or a combination thereof.

In some embodiments, the metabolic disease involves dysregulation ofglucose metabolism. In some embodiments, the metabolic disease isdiabetes. In some embodiments, the metabolic disease is type IIdiabetes. In some embodiments, the metabolic disease is obesity.

In some aspects, a method of lowering the blood glucose or HbA1c levelin a subject in need thereof is provided, comprising administering tothe subject a therapeutically effective amount of the polypeptidedescribed herein or of a pharmaceutical composition of such polypeptide.In some aspects, a method of stabilizing the serum level of insulin in asubject in need thereof is provided, comprising administering to thesubject a therapeutically effective amount of the polypeptide describedherein or of a pharmaceutical composition of such polypeptide. In someembodiments, the subject is suffering from or at risk of developing ametabolic disease involving dysregulation of glucose metabolism. In someembodiments, the subject is suffering or at risk of developing diabetes.In some embodiments, the subject is suffering from or at a risk ofdeveloping type II diabetes. In some embodiments, the subject issuffering from or at a risk of developing obesity.

Without being bound by theory, it is believed that the polypeptidedescribed herein may be capable of treating the metabolic diseases ormodulating the serum level of metabolism-associated molecules in thesubject by bidirectionally regulating NTCP-mediated bile acid uptake inthe subject following administration of the polypeptide to the subject.In some embodiments, when the concentration of the polypeptide describedherein in the blood stream of a subject administered with suchpolypeptide is at or below a certain concentration, bile acid uptake inthe subject is enhanced. In some embodiments, when the concentration ofthe polypeptide described herein in the blood stream of the subject isabove a certain concentration, bile acid uptake in the subject isinhibited. In some embodiments, when the concentration of thepolypeptide described herein in the blood stream of a subjectadministered with such polypeptide is at or below 93 nmol/L, bile aciduptake in the subject is enhanced. In some embodiments, when theconcentration of the polypeptide described herein in the blood stream ofthe subject is above 93 nmol/L, bile acid uptake in the subject isinhibited. For example, when the concentration of the Cmyr-47polypeptide described herein in the blood stream of a subjectadministered with such polypeptide is at or below 500 ng/ml, bile aciduptake in the subject is enhanced. When the concentration of the Cmyr-47polypeptide described herein in the blood stream of the subject is above500 ng/ml, bile acid uptake in the subject is inhibited. In someembodiments, the serum concentration of the polypeptide described hereinmay be measured at least about 10, 20, 40, 60, 90, 120, 180, 240, or 360minutes following the administration. In some embodiments, the serumconcentration of the polypeptide described herein is above 93 nmol/L bycertain time following the administration. In some embodiments, theserum concentration of the polypeptide described herein is at or below93 nmol/L after that time following the administration. For example, theserum concentration of the Cmyr-47 polypeptide described herein is above500 ng/ml by a certain time following the administration. In someembodiments, the serum concentration of the Cmyr-47 polypeptidedescribed herein is at or below 500 ng/ml after that time following theadministration. In some embodiments, such threshold serum concentrationof the polypeptide occurs at about 20 minutes following theadministration.

As used herein, a “metabolic disease” or “metabolic disorder” includesany disease that may be caused by dysregulation of metabolic pathways,such as, e.g., pathways involved in bile acid metabolism, glucosemetabolism, lipid metabolism, and amino acid metabolism. In someembodiments, the metabolic disease refers to a disease that involvesdysregulation of lipid metabolism. In certain embodiments, the metabolicdisease refers to a disease that comprises dysregulation in producing,clearing, and/or utilizing lipid metabolites including, e.g., bileacids, cholesterol, triglycerides, and fatty acids. The metabolicdisease described herein therefore may refer to a cholesterol-relateddisorder. The metabolic disease described herein therefore may refer tohyperlipidemia, such as, e.g., hypertriglyceridemia,hypercholesterolemia, or a combination thereof. In some embodiments, themetabolic disease refers to a disease that involves dysregulation ofglucose metabolism. In certain embodiments, the metabolic disease refersto a disease that comprises dysregulation in producing, clearing, and/orutilizing glucose metabolites, e.g., glucose, pyruvate, andglucose-6-phosphate. The metabolic disease described herein thereforemay refer to diabetes and obesity.

The metabolic disease described herein may be chosen from hyperglycemia;hypoglycemia; hyperinsulinemia; obesity, hyperlipidemia;hypertriglyceridemia; hypercholesterolemia; heart disease; metabolicsyndrome; atherosclerotic disease; coronary heart disease; coronaryartery disease; peripheral arterial disease; angina pectoris;cerebrovascular disease; acute coronary syndrome; myocardial infarction;stroke; cardiovascular disease; Alzheimer's disease; dyslipidemias;familial combined hyperlipidemia; familial hypertriglyceridemia;familial hypercholesterolemia; heterozygous hypercholesterolemia;homozygous hypercholesterolemia; familial defective apolipoproteinB-100; polygenic hypercholesterolemia; remnant removal disease; hepaticlipase deficiency; dyslipidemia caused by dietary indiscretion,hypothyroidism, drugs including estrogen and progestin therapy,beta-blockers, and thiazide diuretics; nephrotic syndrome; chronic renalfailure; Cushing's syndrome; primary biliary cirrhosis; glycogen storagedisease; hepatoma; cholestasis; acromegaly; insulinoma; isolated growthhormone deficiency; kidney impairment; obesity; and alcohol-inducedhypertriglyceridemia.

The term “metabolic disease” includes, e.g., type I diabetes, type IIdiabetes, hyperglycemia, hypoglycemia, hyperinsulinemia, obesity,hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, heartdisease, metabolic syndrome, coronary heart disease, stroke,cardiovascular diseases, Alzheimer's disease, and generallydyslipidemias, which can be manifested, for example, by an elevatedtotal serum cholesterol, LDL, triglycerides, VLDL, and/or HDL. Somenon-limiting examples of primary and secondary dyslipidemias that can betreated using the polypeptides described herein, either alone or incombination with one or more other agents include metabolic syndrome,diabetes, hyperlipidemia, familial combined hyperlipidemia, familialhypertriglyceridemia, familial hypercholesterolemia, includingheterozygous hypercholesterolemia, homozygous hypercholesterolemia,familial defective apolipoprotein B-100; polygenic hypercholesterolemia;remnant removal disease, hepatic lipase deficiency; dyslipidemiasecondary to any of the following: dietary indiscretion, hypothyroidism,drugs including estrogen and progestin therapy, beta-blockers, andthiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing'ssyndrome, primary biliary cirrhosis, glycogen storage diseases,hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormonedeficiency, and alcohol-induced hypertriglyceridemia.

In some embodiments, the polypeptide described herein is useful inpreventing or treating one or more metabolic diseases. In certainembodiments, the polypeptides described herein can also be useful inpreventing or treating one or more symptoms or complications associatedwith a metabolic disease. For instance, the polypeptide can be used toprevent or treat cardiovascular diseases, including atheroscleroticdiseases, such as, e.g., coronary heart disease, coronary arterydisease, peripheral arterial disease, stroke (ischaemic andhemorrhagic), angina pectoris, or cerebrovascular disease and acutecoronary syndrome, myocardial infarction. In certain embodiments, thepolypeptides described herein can also be useful in preventing ortreating heart diseases, kidney impairment, or obesity associated with ametabolic disorder.

The term “diabetes” refers to a disease or condition generallycharacterized by metabolic defects in production and/or utilization ofglucose that result in the failure to maintain appropriate blood sugarlevels in the body. The results of these defects include elevated bloodglucose, referred to as “hyperglycemia.” Two major forms of diabetes areType I diabetes and Type II diabetes. Type I diabetes generally resultsfrom an absolute deficiency of insulin (e.g., the production frompancreatic R cells is extremely low or completely ablated), thereforefailing to regulate glucose utilization. Type II diabetes often occursin the face of normal or even elevated levels of insulin, and can resultfrom the inability of tissues to respond appropriately to insulin. MostType II diabetic patients are insulin resistant and have a relativedeficiency of insulin in that insulin secretion cannot compensate forthe resistance of peripheral tissues to respond to insulin.

The term “hyperlipidemia” refers to a condition characterized by anabnormal increase in serum lipids. The lipids fractions in thecirculating blood include, e.g., total cholesterol, certainlipoproteins, and triglycerides. Serum lipoproteins serve as carriersfor lipids in the circulation and are classified by their density,including: chylomicrons, very low density lipoproteins (“LDL”),intermediate density lipoproteins (“IDL”), low density lipoproteins(“LDL”), and high density lipoproteins (“HDL”). The term“hyperlipidemia” encompasses primary and secondary hyperlipidemia.Primary hyperlipidemia is generally caused by genetic defects, whilesecondary hyperlipidemia is generally caused by other factors, such asvarious disease states, drugs, and dietary factors. For example,secondary hyperlipidemia may be caused by diabetes. Alternatively,hyperlipidemia can result from a combination of primary and secondarycauses. Hyperlipidemia may encompass hypertriglyceridemia,hypercholesterolemia, or a combination thereof. The term“hypertriglyceridemia,” as used herein, refers to a condition in whichserum total triglyceride levels are elevated above a desired level. Theterm “hypercholesterolemia,” as used herein, refers to a condition inwhich serum cholesterol levels are elevated above a desired level. Incertain embodiments, the serum total cholesterol, HDL cholesterol(“HDL-C”), or LDL cholesterol (“LDL-C”) levels are elevated above thedesired level in hypercholesterolemia. Hyperlipidemia also imposes arisk in and may encompass development of cardiovascular andatherosclerosis diseases. The term “cardiovascular disease” encompassesa disease of the blood vessels of the circulation system caused byabnormally high concentrations of lipids in the vessels. The term“atherosclerosis” refers to a disease of the arteries in which fattyplaques develop on the inner walls, with eventual obstruction of bloodflow.

“Patient” and “subject” may be used interchangeably to refer to ananimal, such as a mammal or a human, being treated or assessed for adisease, disorder, or condition, at risk of developing a disease,disorder, or condition, or having or suffering from a disease, disorder,or condition. In some embodiments, such disease, disorder, or conditionmay include a metabolic disease. In some embodiments, the metabolicdisease may involve dysregulation of lipid metabolism. In someembodiments, the metabolic disease may be a cholesterol-relateddisorder, such as hyperlipidemia (e.g., hypercholesterolemia,hypertriglyceridemia, or a combination thereof). In some embodiments,the metabolic disease may involve dysregulation of glucose metabolism.In some embodiments, the metabolic disease may be diabetes, includingtype I and type II diabetes.

The term a “therapeutically effective amount” or “effective amount” of apolypeptide described herein or a composition comprising suchpolypeptide refers to an amount effective in the prevention or treatmentof a disorder for the treatment of which the polypeptide or compositionis effective. The term may include an amount of the polypeptidedescribed herein that is effective in increasing the serum level of bileacid in a subject suffering from or at a risk of developing a metabolicdisease. The term may include an amount of the polypeptide describedherein that is effective in lowering the blood glucose or HbA1c level orstabilizing the serum level of insulin in a subject suffering from or ata risk of developing a metabolic disease, such as, e.g., diabetes andobesity. The term may also include an amount of the polypeptidedescribed herein that is effective in lowering the level of serumlipids, such as, e.g., the total cholesterol, total triglycerides, LDLcholesterol level, in a subject suffering from or at a risk ofdeveloping a metabolic disease, such as, e.g., hyperlipidemia, includinghypercholesterolemia, hypertriglyceridemia, or both. The term alsoincludes an amount of a polypeptide described herein that, whenadministered to a subject for treating a metabolic disease, such as,e.g., diabetes and hyperlipidemia (e.g., hypercholesterolemia,hypertriglyceridemia, or both), is sufficient to effect treatment of thedisease, e.g., by diminishing, ameliorating, or maintaining the existingdisease or one or more symptoms of the disease, or by inhibiting theprogression of the disease. The “therapeutically effective amount” or“effective amount” may vary depending on the polypeptide, the route ofadministration, the disease and its severity, and the health, age,weight, family history, genetic makeup, stage of pathological processes,the types of preceding or concomitant treatments, if any, and otherindividual characteristics of the subject to be treated.

The “therapeutically effective amount” of the polypeptide describedherein may allow the administered polypeptide to reach a concentrationin the blood stream of the subject at which the polypeptide is capableof inhibiting NTCP-mediated bile acid uptake in the subject. In someembodiments, a therapeutically effective amount of the polypeptidedescribed herein may allow the administered polypeptide to reach atleast about 93 nmol/L in the blood stream of a subject administered withthat amount. For example, a therapeutically effective amount of theCmyr-47 polypeptide described herein may allow the administeredpolypeptide to reach at least about 500 ng/ml in the blood stream of asubject administered with that amount.

In certain embodiments, a therapeutically effective amount of thepolypeptide described herein refers to an amount such that the serumconcentrations of the administered polypeptide allow for bidirectionalregulation of NTCP-mediated bile acid uptake in the subject. Forinstance, when a subject is administered with a therapeuticallyeffective amount of the polypeptide described herein, the initial serumconcentration of the polypeptide in the subject may be above a certainconcentration where the polypeptide inhibits NTCP-mediated bile acidsuptake. The serum concentration of the polypeptide in the subject maygradually reduce and fall to or below a value where the polypeptidebegins enhancing NTCP-mediated bile acids uptake. The serumconcentration of the administered polypeptide may be assessed at leastabout 10, 20, 40, 60, 90, 120, 180, 240, or 360 minutes after theadministration. In some embodiments, the therapeutically effectiveamount of the polypeptide described herein allows the polypeptide toreach a serum concentration above 93 nmol/L by certain time followingthe administration. In some embodiments, the therapeutically effectiveamount of the polypeptide described herein allows the polypeptide toreach a serum concentration at or below 93 nmol/L after that certaintime following the administration. For example, the therapeuticallyeffective amount of the Cmyr-47 polypeptide described herein allows thepolypeptide to reach a serum concentration above 500 ng/ml by certaintime following the administration. In some embodiments, thetherapeutically effective amount of the Cmyr-47 polypeptide describedherein allows the polypeptide to reach a serum concentration at or below500 ng/ml after that certain time following the administration. In someembodiments, the therapeutically effective amount of the polypeptidedescribed herein produces such threshold serum concentration of thepolypeptide at about 20 minutes following the administration.

In various embodiments, the term “treatment” includes treatment of asubject (e.g. a mammal, such as a human) or a cell to alter the currentcourse of the subject or cell. Treatment includes, e.g., administrationof a polypeptide described herein or a pharmaceutical compositioncomprising such polypeptide, and may be performed eitherprophylactically or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. Also included are “prophylactic”treatments, which can be directed to reducing the rate of progression ofthe disease or condition being treated, delaying the onset of thatdisease or condition, or reducing the severity of its onset. “Treatment”or “prophylaxis” does not necessarily indicate complete eradication,cure, or prevention of the disease or condition or the associatedsymptoms. In various embodiments, the term “treatment” may includerelieving, slowing, or reversing the pathological processes or symptomsin a subject suffering from a metabolic disease, such as, e.g., diabetesand hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia, orboth). In some embodiments, the term “treatment” may include improvingat least one symptom or measurable parameter of a metabolic disease. Itwill be apparent to one of skill in the art which biological and/orphysiological parameters can be used to access the pathological processof the metabolic disease. Such pathological processes or symptoms mayinclude, e.g., excessive or increased levels compared with healthysubjects of one or more chemical or biological molecules associated withmetabolism, such as, e.g., glucose, triglyceride, cholesterol, freefatty acids, bile acids, amino acids, hormones, including, e.g.,insulin, LDL-C, HDL-C, HbA1c, blood urea nitrogen, and minerals; or ofone or more physiological parameters that measure metabolic changes,such as, e.g., glycemia, blood pressure, body weight, fat mass, bodymass index (BMI), inflammation, atherosclerosis index (AI), heart index,kidney index, total fat index, and homeostatic model assessment (HOMA)index.

The terms “administering,” or “administer” include delivery of thepolypeptide described herein to a subject either by local or systemicadministration. Administration may be topical (including ophthalmic andto mucous membranes including vaginal and rectal delivery), pulmonary(e.g., by inhalation or insufflation of powders or aerosols, includingby nebulizer, intratracheal, intranasal), epidermal, transdermal, oral,or parenteral. Parenteral administration includes intravenous,subcutaneous, intraperitoneal, or intramuscular injection or infusion;or intracranial, e.g., intrathecal or intraventricular, administration.

In certain embodiments, the present disclosure provides use of thepolypeptides described herein in modulating, such as, e.g., reducing orstabilizing, the level or activity of one or more chemical or biologicalmolecules associated with metabolism, such as, e.g., glucose,triglyceride, cholesterol, free fatty acids, bile acids, amino acids,hormones, LDL-C, HDL-C, HbA1c, blood urea nitrogen, and minerals in asubject. In some embodiments, the present disclosure provides use of thepolypeptides described herein in modulating, such as, e.g., reducing orstabilizing, the level or value of one or more physiological parametersthat measure metabolic changes such as, e.g., glycemia, blood pressure,body weight, fat mass, body mass index (BMI), inflammation,atherosclerosis index (AI), heart index, kidney index, total fat index,and homeostatic model assessment (HOMA) index.

In some embodiments, the present disclosure provides use of thepolypeptides described herein in reducing the level of serum lipids in asubject. In some embodiments, the present disclosure provides use of thepolypeptides described herein in reducing the serum level of totalcholesterol in a subject. In further embodiments, the present disclosureprovides use of the polypeptides described herein in reducing the serumlevel of LDL-cholesterol in a subject. In some embodiments, the presentdisclosure provides use of the polypeptides described herein in reducingthe serum level of triglyceride in a subject. In some embodiments, thepresent disclosure provides use of the polypeptides described herein inreducing the serum level of glucose in a subject. In some embodiments,the present disclosure provides use of the polypeptides described hereinin stabilizing the serum level of insulin in a subject. In someembodiments, the subject may be a mammal. In some embodiments, thesubject may be a human.

In some embodiments, the present disclosure provides use of thepolypeptides described herein in reducing the risk of developing ametabolic disease. In certain embodiments, the present disclosure alsoprovides use of the polypeptides described herein in reducing the riskof developing one or more symptoms or complications associated with ametabolic disease. In some embodiments, the metabolic disease involvesdysregulation of lipid metabolism. In some embodiments, the metabolicdisease is a cholesterol-related disorder. In some embodiments, themetabolic disease is hyperlipidemia. The hyperlipidemia may includehypertriglyceridemia, hypercholesterolemia, or both. In someembodiments, the metabolic disease involves dysregulation of glucosemetabolism. In some embodiments, the metabolic disease is diabetes. Thediabetes may include type I and type II diabetes. In some embodiments,the metabolic disease is obesity. The symptoms or complicationsassociated with the metabolic disease may include, e.g., cardiovasculardiseases such as atherosclerosis diseases, heart diseases, kidneyimpairment, or obesity, in a subject having such metabolic disease (suchas, e.g., diabetes, hyperlipidemia, including hypertriglyceridemia,hypercholesterolemia, or both).

The present disclosure also provides methods to carry out the above usesof the polypeptides described herein in a subject. Such methods maycomprise administering to the subject a therapeutically effective amountof a polypeptide described herein or of a pharmaceutical compositioncomprising such polypeptide. In some embodiments, the subject may be amammal. In some embodiments, the subject may be a human. In someembodiments, the subject may suffer from a metabolic disease or may beat risk of developing such disease.

In a further aspect, the present disclosure provides for the use of thepolypeptides described herein in the manufacture or preparation of amedicament. In some embodiments, the medicament may be for treatment ofa metabolic disease. The metabolic disease may involve dysregulation oflipid metabolism. In some embodiments, the medicament may be fortreatment of a cholesterol-related disorder. In some embodiments, thecholesterol-related disorder may be hyperlipidemia. The hyperlipidemiamay be hypertriglyceridemia, hypercholesterolemia, or a combinationthereof. In yet another embodiment, the medicament is for use in amethod of lowering the level of serum lipids, such as, e.g., the totalcholesterol, total triglyceride, or LDL cholesterol level, in a subject,comprising administering to the subject a therapeutically effectiveamount of the medicament.

In some embodiments, the metabolic disease may involve dysregulation ofglucose metabolism. In some embodiments, the medicament may be fortreatment of diabetes e.g., type I or type II diabetes. In someembodiments, the medicament may be for treatment of obesity. In anotherembodiment, the medicament is for use in a method of lowering the bloodglucose or HbA1c level in a subject, comprising administering to thesubject a therapeutically effective amount of the medicament.

In certain embodiments, the disorder treated may be any disease orcondition which can be improved, ameliorated, inhibited, or prevented bybidirectionally regulating NTCP activity. In certain embodiments,disorders or disease that can benefit from the regulation of bile acidintake by hepatocytes can also be treated by the polypeptides describedherein. In certain embodiments, subjects treatable by the polypeptidesand methods and uses described herein may include subjects indicated forLDL apheresis, subjects with diabetes, subjects with primaryhyperlipidemia (including hypercholesterolemia, hypertriglyceridemia, ora combination thereof) who are intolerant or uncontrolled by othertherapeutic agent, and subjects at risk for developing hyperlipidemiawho may be preventably treated. Other indications include dyslipidemiaassociated with secondary causes such as Type 2 diabetes mellitus,cholestatic liver diseases (primary biliary cirrhosis), nephroticsyndrome, hypothyroidism, obesity, and the prevention and treatment ofcardiovascular diseases (e.g., atherosclerotic diseases), heartdiseases, and kidney impairment.

In certain embodiments, the methods and uses described herein mayfurther comprise administering to the subject an effective amount of atleast one additional therapeutic agent. In certain embodiments, theadditional therapeutic agent may be for preventing and/or treating oneor more diseases associated with the metabolic diseases describedherein, such as, e.g., one or more diseases associated with diabetes orhyperlipidemia. In certain embodiments, the additional therapeutic agentmay be for preventing and/or treating cardiovascular diseases (e.g.,atherosclerotic diseases). In certain embodiment, the additionaltherapeutic agent may be for reducing the risk of recurrentcardiovascular events. In certain embodiments, the additionaltherapeutic agent may be for preventing and/or treating heart diseases,kidney impairment, or obesity. The polypeptides described herein can beused either alone or in combination with other agents in a therapy. Forinstance, any of the polypeptides described herein may be administeredbefore, concurrently with, or after administration of at least oneadditional therapeutic agent. In certain embodiments, the additionaltherapeutic agent may be chosen from e.g., an antihyperlipidemic agent,an antihyperglycemic agent, an antidiabetic agent, an antiobesity agent,and a bile acid analogue.

In some embodiment, the antihyperglycemic agent may be chosen from,e.g., a biduanide (e.g., metformin, phenformin, and buformin), insulin(e.g., regular human insulin, NPH insulin, insulin aspart, insulinlispro, insulin glargine, insulin detemir, and insulin levemir), aglucagon-like peptide 1 receptor agonist (GLP-1RA; e.g., albiglutide,dulaglutide, exenatide, liraglutide, lixisenatide, and extended-releaseglucagon), a sodium-glucose cotransporter 2 inhibitor (SGLR2I; e.g.,canagliflozin, empagliflozin, dapagliflozin, empagliflozin, andipragliflozin), a dipeptidyl peptidase 4 inhibitor (DPP4I; e.g.,bromocriptine, sitagliptin, vildagliptin, saxagliptin, linagliptin,anagliptin, teneligliptin, alogliptin, trelagliptin, gemigliptin,dutogliptin, omarigliptin, berberine, and lupeol), an α-glucosidaseinhibitor (AGI; e.g., miglitol, acarbose, and voglibose), athiazolidinedione (TZD; e.g., pioglitazone, rosiglitazone,lobeglitazone, troglitazone, ciglitazone, darglitazone, englitazone,netoglitazone, rivoglitazone, and mifepristone), a meglitinide (e.g.,repaglinide, nateglinide, and mitiglinide), a sulfonylurea (SU; e.g.,carbutamide, acetohexamide, chlorpropamide, tolbutamide, tolazamide,glipizide (glucotrol), gliclazide, glibenclamide, glyburide (e.g.Micronase), glibornuride, gliquidone, glisoxepide, glyclopyramide,glimepiride, amaryl, and glimiprime), an amylin analogue (e.g.,pramlinitide), a proprotein convertase subtilisin/kexin type 9 inhibitor(PCSK9I; e.g., evolocumab, bococizumab, alirocumab, 1D05-IgG2, RG-7652,LY3015014, RNAi therapeutic ALN-PCSO2, AMG-145, and REGN727/SAR236553),a glucokinase activator (GKA; e.g., MK-0941, RO-28-1675, and AZD1656), aPPAR agonist/modulator, a glucagon receptor antagonist, a C-C chemokinereceptor type 2 (CCR2) antagonist, an Interleukin-1 modulator, aG-protein coupled receptor agonist, a gastrointestinal peptide agonistother than GLP-1, an SGLT1 and dual SGLT1/SGLT2 inhibitor (excluding anSGLT2-only inhibitor), an 11beta-HSD1 inhibitor, a diacylglycerolacyltransferase (DGAT)-1 inhibitor, a cannabinoid, a hepatic carnitinepalmitoyltransferase 1 (CPT1) inhibitor, a fibroblast growth factor(FGF)-21 agonist, a glucocorticoid receptor antagonist, a heat shockprotein (HSP) inducer, a melanocortin-4 receptor (MC4R) agonist, atetrahydrotriazin containing oral antidiabetic, glimin, a proteintyrosine phosphatase 1B (PTP1B) inhibitor, a sirtuin1 (SIRT1) activator,and a microbiome modulator.

In some embodiment, the additional therapeutic agent is anantihyperlipidemic agent and may be chosen from, e.g., a statin (e.g.,HMG-CoA reductase inhibitor; e.g., smvastatin, atorvastatin,rosuvastatin, pravastatin, pitavastatin, lovastatin, atorvastatin,fluvastatin, cerivastatin, mevastatin, pantethine, elastase, andprobucol), a fibric acid (e.g., bezafibrate (e.g., Bezalip),ciprofibrate (e.g., Modalim), clofibrate, gemfibrozil (e.g., Lopid),fenofibrate (e.g., TriCor), clinofibrate (e.g., Lipoclin), lifibrate,alufibrate, simfibrate, etofylline clofibrate, and gemfibrozil), anicotinic acid (e.g., niacin, inositol hexanicotinate, nicotinamide, andacipimox), a bile acid sequestrant (e.g., cholestyramine (e.g.,Questran®), colesevelam (e.g., Welchol®), colestipol (e.g., Colestid®),polidexide, dholestyramine, and divistyramine), ezetimibe (e.g., Zetia),a proprotein convertase subtilisin/kexin type 9 inhibitor (PCSK9I; e.g.,evolocumab, bococizumab, alirocumab, 1D05-IgG2, RG-7652, LY3015014, RNAitherapeutic ALN-PCSO2, AMG-145, and REGN727/SAR236553), a microsomaltriglyceride transfer protein inhibitor (MTTPI; e.g., lomitapide andJTT-130), an apolipoprotein B inhibitor (apoBI; e.g., mipomersen (e.g.,Kynamro)), a diacylglycerol acyltransferase 1 (DGAT1) inhibitor (e.g.,pradigastat), an angiopoietin-like protein 3 inhibitor (e.g., REGN1500),a cholesteryl ester transfer protein (CETP) inhibitor (e.g., anacetrapiband evacetrapib), a peroxisome proliferator-activated receptor (PPAR)α/γ agonist, an acyl-CoA inhibitor, an incretin mimetics inhibitor, anangiopoietin-like protein 3 (ANGPTL3) inhibitor, an angiopoietin-likeprotein 4 (ANGPTL4) inhibitor, an apoC-III-targeted inhibitor, and aselective peroxisome proliferator-activated receptor modulator (SPPARM).

In some embodiment, the additional therapeutic agent is an antiobesityagent and may be chosen from, e.g., orlistat (e.g., Xenical), lorcaserin(e.g., Belviq), phentermine, topiramate, diethylpropion,phendimetrazine, benzphetamine, and a combination of phendimetrazine andbenzphetamine.

In some embodiment, the additional therapeutic agent is a bile acidanalogue and may be chosen from, e.g., obeticholic acid, ursodeoxycholicacid, and cholylsarcosine.

In some embodiments, the additional therapeutic agent may also be chosenfrom, e.g., a farnesoid X receptor (FXR) agonist, an FXR inhibitor, atransmembrane G protein-coupled receptor 5 (TGR5) agonist, and a TGR5inhibitor.

In some embodiments, the additional therapeutic agent may be chosen frominsulin, metformin, sitagliptin, colesevelam, glipizide, simvastatin,atorvastatin, ezetimibe, fenofibrate, nicotinic acid, orlistat,lorcaserin, phentermine, topiramate, obeticholic acid, andursodeoxycholic acid.

Such combination therapies described herein may encompass combinedadministration (where two or more therapeutic agents are included in thesame or separate formulations), and separate administration, in whichcase, administration of the polypeptides described herein can occurprior to, simultaneously, and/or following, administration of theadditional therapeutic agent.

The polypeptides described herein (and any additional therapeutic agent)can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment orintralesional administration. In some embodiments, the polypeptidesdescribed herein may be parenterally administered. Parenteraladministration may include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In some embodiments,the polypeptides described herein may administered subcutaneously. Insome embodiments, the polypeptides described herein may administeredintravenously. Dosing can be by any suitable route, such as, e.g., byinjections or infusions, such as intravenous or subcutaneous injectionsor infusions, depending in part on whether the administration is briefor chronic. Various dosing schedules including e.g. single or multipleadministrations over various time-points, bolus administration, andpulse infusion are also contemplated.

The polypeptides described herein would be formulated, dosed, andadministered in a fashion consistent with common medical practice.Factors for consideration in this context may include, e.g., theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The polypeptides described herein neednot be but can be optionally formulated with one or more agentscurrently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of thepolypeptide described herein present in the formulation, the type ofdisorder or treatment, and other factors discussed above.

For the prevention or treatment of disease, the appropriate dosage of apolypeptide described herein (when used alone or in combination with oneor more other additional therapeutic agents) may depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe polypeptide is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to thepolypeptide, and the discretion of the attending physician. Thepolypeptides described herein may be suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, the polypeptide described herein maybe administered to the patient, for example, by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentmay be sustained until a desired suppression of disease symptoms occurs.The polypeptide described herein may be administered intermittently,e.g. every day, every two days, every three days, every week, or everytwo or three weeks (e.g. such that the patient receives from more thanone, such as, e.g., about two to about twenty, or e.g. about six dosesof the polypeptide). An initial higher loading dose, followed by one ormore lower doses, may be administered.

In certain embodiments, a flat-fixed dosing regimen may be used toadminister the polypeptide described herein to a subject. However, otherdosage regimens may also be useful depending on the factors discussedabove. The progress of this therapy can be easily monitored byconventional techniques and assays for the disease or condition treated.

The following Examples may be used for illustrative purposes and shouldnot be deemed to narrow the scope of the invention.

Example 1 Example 1.1. Synthesis of Polypeptides

Polypeptides as shown in Table 1 were synthesized according to thestandard Fmoc protocol for polypeptide synthesis. Generally, individualamino acid residues were extended from the carboxyl terminus to theamino terminus, starting from a MBHA resin. The N terminus was thenmodified by myristoylation. After completion of peptide synthesis, thepolypeptides were cleaved from the resin by a cleavage solution and theC terminus of the polypeptides was further modified by amination. Theresin was removed by filtering with G6 sand-core funnel and the filtratecontaining the polypeptides was dried under vacuum. The polypeptideproduct was dissolved in deionized water, and purified in AKTA explorer100 type medium pressure liquid chromatograph equipped with C18 column.The main peaks were recovered stepwise. The samples collected from thetarget peak were analyzed by Agilent 1100 type reversed phase highpressure liquid chromatography (HPLC) equipped with C18 column for theirpurities and confirmed by mass spectrometry for their molecular weights.The collected solutions purified by medium pressure liquidchromatography were freeze-dried for storage. The dried samples weredissolved in PBS and then filtered through a 0.20 μM membrane. Thepolypeptide stocks dissolved in PBS were stored at −80° C. before use.FIG. 1A shows an exemplary graph depicting the purity of the synthesizedpolypeptide, Cmyr-47, as measured by HPLC. FIG. 1B shows an exemplarygraph confirming the correct molecular weight of Cmyr-47 (5398.8 Da) asmeasured by mass spectrometry.

Example 1.2. Binding Assay of Cmyr-47 and NTCP

To demonstrate that a polypeptide derived from HBV can bind to NTCP,various cell-lines expressing NTCP were treated with Cmyr-47. Tovisualize Cmyr-47, the polypeptide was labeled with FITC. Because NTCPexpresses highly in the liver, primary hepatocytes from tupaia and humanhepatocyte cell line HepG2 cells were prepared for the study. As shownin FIGS. 2A and 2B, FITC labeled Cmyr-47 binds to hepatocytes from twodifferent species (FIG. 2A depicting binding of Cmyr-47 to tupaiaprimary hepatocytes; FIG. 2B depicting binding of Cmyr-47 to HepG2cells). To demonstrate that Cmyr-47 specifically binds to NTCP, NTCPexpressing L02 cells (NTCP-L02) were established by transfecting NTCPexpressing vector. L02 cells transfected with a vector that does notexpress NTCP (BLANK-L02) were used as a negative control. As shown inFIG. 3 , Cmyr-47 labeled with FITC binds to NTCP-L02 cells, but failedto bind to BLANK-L02 cells. To confirm the specificity of Cmyr-47,HEK293 cells, a cell-line that is not derived from the liver, wereprepared for the study. NTCP expressing HEK293 cells (NTCP-293) wereestablished by transfecting the cells with an NTCP expressing vector.HEK293 cells transfected with a control vector that does not expressNTCP (BLANK-293) were used as a negative control. As shown in FIG. 4 ,Cmyr-47 labeled with FITC binds to NTCP-293 cells, but not BLANK-293cells. In contrast, as expected based on a previous finding that anavian HBV does not infect mammals (Gripon et al., J. Virol.79(3):1613-22 (2005)), a control polypeptide of 47 amino acid residuesderived from heron HBV pre-S1 region(myristoylated-GLNQSTFNPLGFFPSHQLDPLFKANAGSADWDKNPNKDPWPQAHDTA-amidated, SEQ ID NO: 49) failed to bind to NTCP-293 cells. Theseresults demonstrate that Cmyr-47 specifically binds to NTCP.

Example 1.3. In Vitro Bile Acids Assay of Cmyr-47

To further study the effect of the polypeptide derived from HBV on bileacids transport, NTCP-293 cells were incubated with bile acids labeledwith ³H (³H-TA; taurocholate). By using ³H-TA, the amount of bile acidsabsorbed by the cells can be quantified by measuring the level ofradioactivity of the cells. Since HEK293 cells are not capable ofabsorbing bile acids without NTCP expression, any bile acids taken up byNTCP-293 cells can be contributed to NTCP. TA at 10 umol/L(radioactively labeled as ³H-TA at 0.5 uCi/ml) and increasing amounts ofCmyr-47 were simultaneously added to cultured cells for 10 minutes.Cyclosporine A (CsA), known to inhibit bile acids transport, was used asa positive control at a concentration of 50 μM. As shown in FIG. 5A,compared with BLANK-293 cells, NTCP-293 cells were capable of absorbinga significant amount of bile acids. When NTCP-293 cells were treatedwith Cmyr-47, the uptake of bile acids was bidirectionally regulated atdifferent concentrations of Cmyr-47. As shown in FIG. 5B, Cmyr-47enhanced NTCP transport of bile acids into hepatocytes at aconcentration at and below 500 ng/ml, while Cmyr-47 significantlyreduced TA absorption at a concentration more than 500 ng/ml. Notably,62.5 ng/ml of Cmyr-47 increased TA absorption by more than 50%, while 2μg/ml of Cmyr-47 effectively inhibited the uptake of bile acids ascompared with the positive control, CsA. Based on the escalating dosestudy shown in FIG. 5B, Cmyr-47 was determined to have an IC₅₀ value of0.15 μM in blocking bile acids transport.

In contrast to bidirectional regulation of NTCP-mediated transport ofbile acids by Cmyr-47, one-way inhibition of TA absorption was inducedby CsA (FIGS. 5C and 5D). CsA did not enhance NTCP-mediatedtransportation of TA at either a low concentration of 10 ng/ml or a highconcentration of 100 μg/ml. Based on the escalating dose study shown inFIG. 5D, the calculated IC₅₀ value of CsA for inhibiting bile acidstransport was 3.05 M.

Example 1.4. In Vitro Bile Acids Assay of Additional HBV-DerivedPolypeptides

To further analyze the bidirectional effect of HBV-derived polypeptideson NTCP-mediated uptake of bile acids, two different concentrations(62.5 ng/ml as a representative low concentration and 1 μg/ml as arepresentative high concentration) of Cmyr-47 and other HBV-derivedpolypeptides were tested in the in vitro bile acids assay by followingthe protocol described above. Two control treatments, CsA and quinidine,were also tested for comparison. Quinidine is a class I antiarrhythmicagent and was previously shown to enhance NTCP-mediated TA uptake (seeKim et al, J. Pharmacol. Exp. Ther. 291(3): 1204-09 (1999)). CsA andquinidine were purchased from Sigma-Aldrich (Sigma-30024 andSigma-Q3625, respectively).

As shown in FIG. 5E, CsA at 100 ng/ml had no significant effect on TAabsorption, while quinidine enhanced TA absorption at 0.1 μmol/L.HBV-derived polypeptides listed in Table 1 (Cmyr-60, Cmyr-55, Cmyr-47,Cmyr-40, Cmyr-35, Cmyr-30, Cmyr-25, Cmyr-20, Cmyr-47+(−10),Cmyr-47+(−9), Cplam-47, Cstea-47, Cchol-47, Amyr-47, Bmyr-47, Dmyr-47,Emyr-47, Fmyr-47, Gmyr-47 or Hmyr-47) enhanced TA absorption at a lowconcentration molarly equivalent to 62.5 ng/ml (11.58 nmol/L) ofCmyr-47, confirming that HBV-derived polypeptides are capable ofenhancing NTCP-mediated uptake of bile acids at a low concentration.

In contrast, when HBV-derived polypeptides (Cmyr-60, Cmyr-55, Cmyr-47,Cmyr-40, Cmyr-35, Cmyr-30, Cmyr-25, Cmyr-20, Cmyr-47+(−10),Cmyr-47+(−9), Cplam-47, Cstea-47, Cchol-47, Amyr-47, Bmyr-47, Dmyr-47,Emyr-47, Fmyr-47, Gmyr-47 or Hmyr-47) were tested at a higherconcentration molarly equivalent to 1 μg/ml (185.23 nmol/L) of Cmyr-47,all the polypeptides effectively inhibited TA absorption (FIG. 5F),confirming that HBV-derived polypeptides are capable of inhibitingNTCP-mediated uptake of bile acids at a high certain concentration. Forcomparison, CsA at 50 μg/ml inhibited TA absorption, while quinidine at20 μmol/L enhanced TA absorption. These results confirm that HBV-derivedpolypeptides are capable of bidirectionally regulating NTCP-mediateduptake of bile acids in a dose-dependent manner.

Example 2 Example 2.1. Toxicity and Bile Acids, Total Cholesterol,Triglyceride, and Glycemia Analysis of Rats Treated with Cmyr-47

To determine the toxicity of Cmyr-47 and confirm that Cmyr-47 canregulate bile acids uptake in vivo, 190 Sprague Dawley rats with equalnumber of males and females were subjected to a 6-month chronic toxicitytest. Each rat was subcutaneously injected daily with either PBS as acontrol or Cmyr-47 of 1, 3, or 9 mg/kg for 180 days. The experimentalscheme is shown in Table 2.

TABLE 2 Experimental Design Scheme of 180-Day Long Chronic Toxicity Testin Rats Dosage/injection Concentration Clinic equivalent Rats number (n)Group (mg · kg⁻¹) (mg · ml⁻¹) dosage multiplied by ♀ ♂ I. Control 0 0 020 20 II. Low-dose 1 0.4 2.3 20 + 5 20 + 5 III. Middle-dose 3 1.2 7.020 + 5 20 + 5 IV. High-dose 9 3.6 20.9 20 + 5 20 + 5

At the end of the experiment, a blood sample from each rat was collectedby tail-bleeding. Serums from the blood samples were further separatedout by centrifugation at 3,000 rpm for 10 min. The serum samples werethen analyzed a total bile acid assay kit (Nanjing JianchengBioengineering Institute) in order to determine the level of total bileacids (TBA). As shown in Tables 3 and 4, both female and male ratstreated with Cmyr-47 displayed an increased level of total bile acids inthe serum as compared with the control group, indicating that Cmyr-47 iscapable of effectively blocking the bile acids uptake in vivo. Theincrease of total bile acids was dose-dependent.

TABLE 3 Serum TBA Concentrations of 180-Day Long Chronic Toxicity Testin Rats (Male, μM/L) 95% Confidence Interval for Mean Std. Upper Group NMean Deviation Std. Error Lower Bound Bound Minimum Maximum Control 107.920 3.5401 1.5832 3.524 12.316 5.2 14.0 Low-dose 10 15.480 6.59752.9505 7.288 23.672 9.5 23.6 Middle-dose 10 17.240 8.3575 3.7376 6.86327.617 11.1 30.9 High-dose 10 20.640 11.5881 5.1823 6.252 35.028 13.541.2

TABLE 4 Serum TBA Concentrations of 180-Day Long Chronic Toxicity Testin Rats (Female, μM/L) 95% Confidence Interval for Mean Lower UpperGroup N Mean Std. Deviation Std. Error Bound Bound Minimum MaximumControl 10 13.180 3.4354 1.5364 8.914 17.446 7.8 16.8 Low-dose 10 16.4605.1189 2.2892 10.104 22.816 11.1 22.5 Middle-dose 10 21.860 7.54773.3754 12.488 31.232 12.9 32.7 High-dose 10 26.800 15.4932 6.9288 7.56346.037 13.8 51.0

Example 2.2. Total Cholesterol, Triglyceride, and Glycemia of RatsTreated with Cmyr-47

The levels of total cholesterol (TC), triglyceride (TG), and glycemia(GLU) of each animal tested in Example 2.1 are summarized in Tables 5and 6. As shown in Tables 5 and 6, the low dose, middle dose, or highdose of Cmyr-47 had no significant effect on serum TC, TG or GLU, ascompared with the control group (all values of P>0.05). Therefore,Cmyr-47 does not affect normal levels of serum TC, TG or GLU atphysiological conditions.

TABLE 5 Serum TC, TG, GLU Concentrations of 180-Day Long ChronicToxicity Test in Rats (Male, mmol/L, χ ± SEM) Group n TC TG GLU Control10 1.62 ± 0.37 0.56 ± 0.20 7.15 ± 1.74 Low-dose 10 1.58 ± 0.21 0.61 ±0.26 6.89 ± 1.24 middle-dose 10  1.5 ± 0.18 0.49 ± 0.18 6.76 ± 1.05High-dose 10 1.58 ± 0.33 0.56 ± 0.26 6.69 ± 0.53 Note: compared withnormal control group, ^(#)P < 0.05, ^(##)P < 0.01; compared with modelcontrol group, *P < 0.05, **P < 0.01

TABLE 6 Serum TC, TG, GLU Concentrations of 180-Day Long ChronicToxicity Test in Rats (Female, mmol/L, χ ± SEM) Group n TC TG GLUControl 10 2.28 ± 0.54 0.78 ± 0.25 6.41 ± 0.38 Low-dose 10 1.97 ± 0.360.68 ± 0.33 6.34 ± 0.55 middle-dose 10 1.98 ± 0.49 0.75 ± 0.29 6.17 ±0.69 High-dose 10 1.80 ± 0.50 0.56 ± 0.21 6.11 ± 0.42 Note: comparedwith normal control group, ^(#)P < 0.05, ^(##)P < 0.01; compared withmodel control group, *P < 0.05, **P < 0.01

Example 2.3. Toxicity and Bile Acids, Total Cholesterol, Triglyceride,and Glycemia Analysis of Dogs Treated with Cmyr-47

To further determine the toxicity of Cmyr-47 and confirm whether Cmyr-47can regulate bile acids uptake in vivo, 56 Beagle dogs, with equalnumber of males and females, were subjected to a 9-month chronictoxicity test. Each dog was subcutaneously injected daily with eitherPBS as a control or Cmyr-47 of 0.25, 0.75, or 2 mg/kg per injection for270 days. The experimental scheme is shown in Table 7.

TABLE 7 Experimental Design Scheme of 270- day Chronic Toxicity Test inDogs Clinic Dosage/ equivalent Dogs number injection dosage (n) Group(mg · kg⁻¹) multiplied by ♀ ♂ I. Control 0 0 7 7 II. Low-dose 0.25 2 7 7III. Middle-dose 0.75 6 7 7 IV. High-dose 2 16 7 7

At the end of the experiment, a blood sample from each dog was collectedby vein puncture, and serum from the collected blood samples wasseparated as described above. The serum samples were then analyzed todetermine the concentration of total bile acids levels as describedabove. As shown in Table 8, Cmyr-47 increased the serum level of totalbile acids in dogs in a dose-dependent manner as compared with thecontrol group. The levels of total cholesterol (TC), triglyceride (TG),and glycemia (GLU) of each animal are summarized in Table 9. As shown inTable 9, the low dose, middle dose, or high dose of Cmyr-47 had nosignificant effect on serum TC, TG or GLU, as compared with the controlgroup (all values of P>0.05). Therefore, Cmyr-47 does not affect normallevels of serum TC, TG or GLU at physiological conditions.

TABLE 8 Serum TBA Concentration of 270-day Long Chronic Toxicity Test inDogs for Day 91 Phased Detection (μM/L) Group Animal Low- Middle- High-Gender No. control dose dose dose Female 1 2.6 3.2 2.4 2.1 7 1.2 3.7 1.01.1 3 1.0 7.8 1.0 20.4 4 0.9 2.2 1.8 13.2 5 1.0 2.3 4.1 4.2 6 2.1 22.32.3 1.4 7 1.6 3.0 2.9 7.3 Male 8 1.4 1.8 1.9 1.2 9 1.7 1.7 2.1 1.7 101.4 3.2 3.7 3.0 11 0.8 2.9 1.2 1.3 12 1.3 1.6 2.4 3.0 13 0.8 1.1 15.35.9 14 1.9 1.2 2.1 2.4 x ± s 1.41 ± 4.14 ± 3.16 ± 4.87 ± 0.53 5.48 3.615.56 P value — 0.086 0.084 0.037

TABLE 9 Serum TC, TG, GLU Concentration of 270-day Long Chronic ToxicityTest in Dogs for Day 91 Phased Detection (mmol/L, χ ± SEM) Group n TC TGGLU Control 14 2.93 ± 0.66 0.39 ± 0.08 4.50 ± 0.68 Low-dose 14 3.07 ±0.44 0.49 ± 0.13 4.26 ± 0.51 middle-dose 14 3.20 ± 0.61 0.45 ± 0.10 4.52± 0.41 High-dose 14 3.16 ± 0.36 0.46 ± 0.12 4.69 ± 0.41 Note: comparedwith normal control group, ^(#)P < 0.05, ^(##)P < 0.01; compared withmodel control group, *P < 0.05, **P < 0.01

Example 2.4. Pharmacokinetic Analysis of Dogs Treated with Cmyr-47

Fifty-six Beagle dogs, with equal number of males and females, weresubjected to a 1-month chronic toxicity test. Each dog wassubcutaneously injected every day with either PBS as a control orCmyr-47 of 0.4, 1.2, or 3.6 mg/kg per injection for 30 days. Theexperimental scheme is shown in Table 10.

TABLE 10 Experimental Design Scheme of Pharmacokinetic Analysis in DogsDosage/injection Dogs number (n) Group (mg · kg⁻¹) ♀ ♂ I. Control 0 3 3II. Low-dose 0.4 3 3 III. Middle-dose 1.2 3 3 IV. High-dose 3.6 3 3

During the experiment, blood samples from each dog was collected by veinpuncture at 0, 10, 20, 40, 60, 90, 120, 180, 240, 360, 1440 min afterthe first dose and last dose. The serum from the collected blood sampleswas separated as described above. The serum samples were then analyzedto determine the concentration of Cmyr-47 by radioimmunoassay (RIA)using anti-PreS1 antibody 125E11 (Wei et al., Clinica. Chimica. Acta.317:159-69 (2002)). Tables 11-13 show the serum concentrations of eachanimal administered with 0.4, 1.2, or 3.6 mg/kg, respectively. As shownin Table 13, dogs administered with 3.6 mg/kg of Cmyr-47 well toleratedthe dosage and no serious toxicity was observed. The peak concentration(i.e. C_(max)) of Cmyr-47 in blood stream was reached at 20 minutes(i.e. T_(max)) following the administration. A C_(max) refers to thepeak serum concentration that a drug achieves after a dosing. A T_(max)refers to the time at which the C_(max) is observed. As shown in Table13, for instance, the highest dose of Cmyr-47 was able to reach theC_(max) of above 500 ng/ml at 20 minutes, indicating the T_(max) ofCymr-47 is about 20 minutes in this test condition. As shown in Tables11-13, only the highest dose, 3.6 mg/kg, was able to reach the peakconcentration above 500 ng/ml.

TABLE 11 The serum Cmyr-47 concentration in Beagle dogs with sc 0.4 mg ·kg − 1 Time post dose Serum concentration of Cmyr-47 (ng · mL⁻¹) min L7L8 L9 L10 L11 L12 MEAN ± SD CV % FIRST 0 ND ND ND ND ND ND — — DOSE 1086.9 91.3 82.7 103.1 126.5 123.2 102.3 ± 18.8 18.4 20 165.2 142.7 116.7162.1 155.8 137.2 146.6 ± 18.3 12.5 40 108.9 100.0 78.6 112.2 97.4 108.4100.9 ± 12.3 12.2 60 50.5 60.2 48.1 62.0 47.5 69.5 56.3 ± 8.9 15.9 9034.6 31.1 37.4 32.3 36.4 30.3 33.7 ± 2.9 8.7 120 23.3 21.6 24.4 21.624.2 19.7 22.5 ± 1.8 8.1 180 11.6 12.9 13.3 11.1 10.9 13.9 12.3 ± 1.210.1 240 2.4 1.5 2.3 2.0 2.6 1.9  2.1 ± 0.4 17.7 360 0.62 0.59 0.33 0.430.31 0.72  0.50 ± 0.17 33.7 1440 ND ND ND ND ND ND — — LAST 0 ND ND NDND ND ND — — DOSE 10 98.8 76.5 81.0 75.3 124.3 78.9  89.1 ± 19.2 21.6 20139.0 127.3 134.0 180.1 146.5 117.7 140.8 ± 21.7 15.4 40 95.6 112.2 90.4100.8 137.2 96.3 105.4 ± 17.2 16.3 60 59.0 47.5 56.9 50.7 56.8 58.9 55.0± 4.8 8.6 90 45.0 24.8 22.7 25.8 38.5 29.9 31.1 ± 8.8 28.4 120 20.9 21.626.0 19.4 25.8 17.4 21.8 ± 3.5 15.8 180 14.3 9.9 13.2 8.7 8.2 10.9 10.8± 2.5 22.7 240 2.3 2.0 2.9 2.9 2.7 2.0  2.5 ± 0.4 16.8 360 0.59 0.530.34 0.39 0.35 0.61  0.47 ± 0.12 26.3 1440 ND ND ND ND ND ND — — Note:ND = undetectable

TABLE 12 The serum Cmyr-47 concentration in Beagle dogs with sc 1.2 mg ·kg⁻¹ Time post dose Serum concentration of Cmyr-47 (ng · mL⁻¹) min M13M14 M15 M16 M17 M18 MEAN ± SD CV % FIRST 0 ND ND ND ND ND ND — — DOSE 10289.9 340.3 249.3 374.1 272.9 327.2 308.9 ± 46.4 15.0 20 433.6 450.2365.0 540.9 457.4 422.1 444.9 ± 57.3 12.9 40 333.9 323.0 259.9 314.9287.1 292.8 302.0 ± 27.2 9.0 60 140.9 166.6 180.7 127.3 123.3 198.5156.2 ± 30.5 19.5 90 90.6 108.9 113.2 82.4 78.9 122.6  99.4 ± 17.9 18.0120 57.2 67.4 54.3 70.9 50.2 61.0 60.2 ± 7.9 13.1 180 41.8 35.2 31.638.6 33.9 49.0 38.3 ± 6.4 16.6 240 5.6 2.2 7.0 7.9 4.5 6.6  5.6 ± 2.136.7 360 2.0 1.6 1.8 2.2 1.6 1.9  1.8 ± 0.2 13.5 1440 ND ND ND ND ND ND— — LAST 0 ND ND ND ND ND ND — — DOSE 10 300.6 301.8 235.7 335.3 305.4344.9 304.0 ± 38.3 12.6 20 374.1 418.2 327.2 492.8 407.6 392.8 402.1 ±54.8 13.6 40 300.9 279.6 235.7 283.7 266.3 259.9 271.0 ± 22.4 8.3 60128.1 149.5 162.1 112.5 109.0 170.9 138.7 ± 26.0 18.8 90 79.1 90.9 90.571.6 68.6 108.3  84.8 ± 14.8 17.4 120 51.0 59.9 48.3 63.4 45.0 54.3 53.6± 7.0 13.1 180 37.4 30.5 27.2 34.8 29.0 41.8 33.4 ± 5.6 16.7 240 3.3 4.75.7 6.9 5.1 4.5  5.0 ± 1.2 23.7 360 1.7 1.4 1.6 2.0 1.4 1.7  1.6 ± 0.213.4 1440 ND ND ND ND ND ND — — Note: ND = undetectable

TABLE 13 The serum Cmyr-47 concentration in Beagle dogs with sc 3.6 mg ·kg − 1 Time post dose Serum concentration of Cmyr-47 (ng · mL⁻¹) min H19H20 H21 H22 H23 H24 MEAN ± SD CV % FIRST 0 ND ND ND ND ND ND — — DOSE 10617.8 510.6 739.4 542.7 569.9 697.1 612.9 ± 89.8  14.7 20 958.4 739.4698.7 1117.3 1206.0 849.6 928.2 ± 204.1 22.0 40 810.7 620.0 739.4 646.3840.6 543.6 700.1 ± 116.0 16.6 60 305.4 344.9 340.4 382.7 425.0 286.4347.5 ± 50.6  14.6 90 218.9 237.5 262.8 201.9 190.3 187.4 216.5 ± 29.5 13.6 120 132.9 109.0 156.0 117.0 148.5 90.7 125.7 ± 124.8 19.7 180 68.544.5 83.2 56.9 75.4 66.0 65.8 ± 13.7 20.8 240 12.8 16.2 11.2 15.0 10.113.2 13.1 ± 2.3  17.4 360 3.1 3.3 2.6 3.5 3.0 3.1 3.1 ± 0.3 9.6 1440 NDND ND ND ND ND — — LAST 0 ND ND ND ND ND ND — — DOSE 10 543.6 433.2635.1 490.3 501.2 588.1 531.9 ± 72.6  13.6 20 874.7 673.9 620.0 921.71129.9 746.1 827.7 ± 187.5 22.7 40 730.7 545.5 674.4 562.1 746.4 493.1625.4 ± 105.9 16.9 60 272.3 293.4 272.8 343.2 394.0 252.8 304.7 ± 53.6 17.6 90 195.9 209.4 236.5 173.7 197.5 166.2 196.5 ± 25.3  12.9 120 112.592.2 132.9 104.5 127.9 97.2 111.2 ± 16.4  14.8 180 53.6 25.9 72.2 56.070.3 53.6 55.3 ± 16.7 30.1 240 10.9 13.8 10.0 13.2 8.6 11.7 11.4 ± 1.9 17.0 360 2.4 3.6 2.4 3.2 3.2 2.8 2.9 ± 0.5 16.3 1440 ND ND ND ND ND ND —— Note: ND = undetectable

Example 3 Example 3.1. Methods and Materials in Treatment ofHyperlipidemic Golden Hamster Model with Cmyr-47

To analyze the in vivo effects of Cmyr-47 on lipid metabolism, ahyperlipidemic golden hamster model was established by feeding hamsterswith a high-fat diet for 2 weeks (after adaptive feeding for 10 days).Male Golden hamsters (N=70, 90-110 g) were purchased from Beijing VitalRiver (SCXK (Jing) 2012-0001) with animal quality certificate number11400700093338. Animals were housed at 23±1° C. with 50-70% humidityunder a 12 hour light:dark cycle (150-200 Lx), in a noise-controlledroom (<50 dB) at the Zhejiang Traditional Chinese Medicine UniversityAnimal Experimental Research Center (SYXK (Zhe) 2013-0184). Thecomposition of the high-fat diet included 1.25% cholesterol and 20.06%fat (soybean oil 2.79%, cocoa butter 17.27%) and the diet was purchasedfrom Research Diets Inc. (New Brunswick, NJ) and stored at 4° C. As anormal non-hyperlipidemic control (“a normal control group”), a group ofgolden hamsters was fed with a regular chow diet. A full nutritional ratpellet was used as a normal chow diet after sterilization with Co⁶⁰irradiation. Animals were provided with filtered and sterilized tapwater ad libitum. All food was available ad libitum and animals werehoused 4 to 5 per cage. Weight and food consumption of each hamster fedwith the high-fat diet was monitored on a weekly basis. All animals weretreated humanely and care was taken to minimize pain and suffering inaccordance with the principle of the 3Rs (replacement, reduction, andrefinement).

After 2 weeks of the high-fat diet treatment, the hyperlipidemicphenotype of hamsters was confirmed when the animals had serum totalcholesterol (TC) levels higher than 10 mmol/L. A total of 40hyperlipidemic hamsters were randomly stratified into 5 groups(N=8/group): a model control group (10 mL/kg PBS, subcutaneousadministration (sc)), a positive treatment control group (fenofibrate,50 mg/kg/day, peroral intragastrical administration (po)), a low-dosetreatment group (Cmyr-47, 10 mg/kg/day, sc), a high-dose treatment group(30 mg/kg/day Cmyr-47, sc), and a CsA treatment group (CsA, 5 mg/kg/day,po). 10 hamsters fed with the normal chow diet were used as a normalcontrol group (10 mL/kg PBS, sc).

By following the protocol described above, Cmyr-47 was synthesized as awhite powder by HEP Pharmaceutical (Shanghai, China; Lot number:14011801). For each treatment, Cmyr-47 was freshly prepared in PBS andused immediately after preparation. PBS was prepared by formulating a20×PB solution (Na₂HPO₄·12H₂O (64.4652 g) and NaH₂PO₄·2H₂O (3.1202 g) inwater to 500 mL). Then, 1 volume of 20×PB, 4 volumes of pure water, and15 volumes of 0.9% normal saline were mixed together to obtain PBS fordissolving the test drug. Fenofibrate (FENO) of Fenolip tablet used as apositive control in this study was purchased from Laboratoires FOURNIERS.A. CsA (Sandimmune®) was purchased from Novartis.

PBS, Cmyr-47, FENO, and CsA were respectively administered for 4consecutive weeks. During the experiment, all animals in the modelcontrol group, low and high dose group, and positive control group werefed with the high fat diet while the normal control group was fed withthe normal chow diet. Cmyr-47 or PBS was subcutaneously injected to thehamsters twice a day (9:00-10:00 in the morning and 16:00-17:00 in theafternoon). A positive treatment control group was provided with FENOvia peroral intragastrical administration. A CsA treatment group wasprovided with CsA via peroral intragastrical administration. Eachmorning, food and water consumption, feces, and animal grooming weremonitored. All animals were weighed weekly.

After 2 weeks of treatment, serum TC and triglycerides (TG) levels ofall animals were measured. After 4 weeks of treatment, serum TC, TG,LDL-C, and HDL-C levels of all animals were measured. Prior tomeasurement, all animals were fasted for 12 h with an access to waterprior to blood collection. A blood (0.3 mL) sample from each animal wasobtained via retro-orbital plexus and serums from the samples werefurther isolated by centrifugation at 3,000 rpm for 10 min. Kits formeasuring lipids were purchased from Shenneng DESAY DiagnosticTechnology Co. Ltd. (Shanghai, China) and all measurements wereconducted by following the manufacturer's protocols. 7020 automaticbiochemical analyzer was used for the measurements. An atherosclerosisindex (AI) of each animal was calculated by following the formula ofAI=(TC−HDL-C)/HDL-C. The AI is considered as one of the most reliableindicators of an increased risk developing atherosclerosis.

An SQP Electronic scale from Sartorius Scientific Instruments Co., Ltd.(Beijing, China) was used, as was a MLS-3750 high pressure sterilizingchamber from Sanyo Company (Japan). An RO-MB-50 Ultrapure water systemwas from Yongjieda Purification Technology Co. Ltd. (Hangzhou, Zhejiang,China). KQ-300DE ultrasound was purchased from Kunshan UltrasonicInstrument Co., Ltd. (Kunshan, Jiangsu, China) and a Hitachi 7020automatic biochemical analyzer from Hitachi Ltd. (Japan). Themultifunctional ELISA machine was from Thermo Fisher Scientific Inc.(USA).

SPSS19.0 software was used to analyze data, expressed as means±standarddeviation or mean±SEM. ANOVA variance analysis was used to evaluate thedata from the test results. LSD test was used for pairwise comparisons.Values of the statistical analyses were rounded to 2 decimal places.

Example 3.2. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum Total Cholesterol (TC)

As shown in Table 14 and FIG. 6 , the serum TC levels in the modelcontrols were significantly higher than those in the normal controlsduring the experiment, confirming that the animals fed with the high-fatdiet were hyperlipidemic (all values of P<0.01). The administration ofFENO significantly lowered the serum TC levels as compared with themodel control group (all of P values less than 0.01). Hyperlipidemicanimals treated with Cmyr-47 also displayed lower serum TC levels thanthe model control group, and the effect of Cmyr-47 was dose-dependent.In contrast, hyperlipidemic animals treated with CsA showedsignificantly elevated serum TC levels as compared with the modelcontrol group.

TABLE 14 Effect of Cmyr-47 on Serum TC (mmol/L, χ ± S) TC level afterweeks of dosing Group Dosage/injection n Before dosing 2 w 4 w Normalcontrol 10 mL/kg PBS 10  4.25 ± 0.41 4.09 ± 0.34 4.39 ± 0.43 Modelcontrol 10 mL/kg PBS 8  11.12 ± 0.47^(##)  19.67 ± 4.05^(##)  19.58 ±4.72^(##) Positive control 50 mg/kg FENO 8 11.34 ± 0.50  6.63 ± 4.40** 7.42 ± 1.70** Low-dose 10 mg/kg Cmyr-47 8 11.10 ± 0.44 15.53 ± 4.51*16.00 ± 5.17* High-dose 30 mg/kg Cmyr-47 8 11.10 ± 0.46  12.67 ± 2.98** 11.54 ± 1.67** CsA treatment  5 mg/kg CsA 8 11.24 ± 0.40 22.53 ± 2.50 24.46 ± 3.35* Note: compared with normal control group, ^(#)P < 0 05,^(##)P < 0.01; compared with model control group, *P < 0.05, **P < 0.01

Additional polypeptides derived from HBV listed in Table 1 were alsotested to confirm their effects on serum TC in vivo. The experiment wasconducted by following the same experimental protocol described above.All polypeptide was administrated at a dose molarly equivalent to 30mg/kg/day of Cmyr-47.

FIGS. 7A and 7B confirmed that animals fed with the high-fat diet werehyperlipidemic (compare the serum TC levels of normal controls and modelcontrols). As previously shown, FENO was capable of decreasing the serumTC levels in the animals. At week 4, animals treated with otherHBV-derived peptides (Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35, Cmyr-30,Cmyr-25, Cmyr-20, Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47, Cstea-47,Cchol-47, Amyr-47, Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47 orHmyr-47) also showed lower serum TC levels than that of the modelcontrols.

Example 3.3. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum Triglycerides (TG)

As shown in Table 15 and FIG. 8 , the serum TG levels in the modelcontrols were significantly higher than the normal controls throughoutthe experiment (all values of P<0.01). Measurements at week 2 and week 4confirmed that FENO significantly decreased the serum TG levels in theanimals (all of P values less than 0.01). The high dose of Cmyr-47 alsosignificantly lowered the serum TG levels in the animals as comparedwith the model controls. Similar to the effect on serum TC, the effectof Cmyr-47 on serum TG were also dose-dependent. Of note, CsA treatmentincreased the serum TG levels in the animals as compared with the modelcontrols.

TABLE 15 Effect of Cmyr-47 on Serum TG (mmol/L, χ ± S) TG level afterweeks of dosing Group Dosage/injection n Before dosing 2 w 4 w Normalcontrol 10 mL/kg PBS 10 2.85 ± 1.24 2.23 ± 0.76 2.29 ± 0.84  Modelcontrol 10 mL/kg PBS 8  5.65 ± 1.78^(##)  7.81 ± 3.24^(##)  7.54 ±3.10^(##) Positive Control 50 mg/kg FENO 8 6.16 ± 1.30  3.14 ± 0.4** 2.68 ± 0.30** Low-dose 10 mg/kg Cmyr-47 8 6.15 ± 1.14 6.29 ± 2.48 4.39± 1.21  High-dose 30 mg/kg Cmyr-47 8 6.35 ± 2.00  5.31 ± 1.76* 3.81 ±0.95* CsA treatment  5 mg/kg CsA 8 6.23 ± 0.90 8.81 ± 0.50 10.23 ±0.61** Note: compared with normal control, ^(#)P < 0.05, ^(##)P < 0.01;compared with model control, *P < 0.05, **P < 0.01

The serum TG levels of animals treated with additional polypeptidesderived from HBV (Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35, Cmyr-30, Cmyr-25,Cmyr-20, Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47, Cstea-47, Cchol-47,Amyr-47, Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47 or Hmyr-47) werealso measured by following the protocol described above. As shown inFIGS. 9A and 9B, the serum TG levels of animals treated with theseHBV-derived polypeptides were lower than that of the model controls,indicating all the tested polypeptides derived from HBV are capable oflowering serum TG in vivo.

Example 3.4. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum LDL-C

Table 16 and FIG. 10 summarize the measurements of serum LDL-C levelsfrom the animals treated with PBS, FENO, or Cmyr-47. As expected, theserum LDL-C levels in the model controls significantly increased ascompared with the normal controls, confirming the hyperlipidemicphenotype (P<0.01). The positive controls treated with FENO displayedsignificantly lower serum LDL-C levels than the model controls.Consistent with the effect of Cmyr-47 on serum TC and TG, animalstreated with Cmyr-47 (particularly with the high dose of 30 mg/kg)showed significantly decreased serum LDL-C levels as compared with themodel controls.

TABLE 16 Effect of Cmyr-47 on Serum LDL-C (4 weeks post the treatment)LDL-C Group Dosage/injection n (mmol/L) Normal control 10 mL/kg PBS 101.09 ± 0.11  Model control 10 mL/kg PBS 8 9.38 ± 3.75^(##)  PositiveControl 50 mg/kg FENO 8 1.88 ± 0.40** Low-dose 10 mg/kg Cmyr-47 8 7.33 ±3.40  High-dose 30 mg/kg Cmyr-47 8 4.34 ± 1.36** Note: compared withnormal control, ^(#)P < 0.05, ^(##)P < 0.01; compared with modelcontrol, *P < 0.05, **P < 0.01

The serum LDL-C levels of animals treated with additional polypeptidesderived from HBV (Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35, Cmyr-30, Cmyr-25,Cmyr-20, Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47, Cstea-47, Cchol-47,Amyr-47, Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47 or Hmyr-47) werealso measured by following the protocol described above. As shown inFIGS. 11A and 11B, the serum LDL-C levels of those animals were lowerthan that of the model controls.

Example 3.5. Effect of Cmyr-47 on Serum HDL-C and AI

Table 17 and FIG. 12 provide the measurements of serum HDL-C levels.Based on the values obtained in this study, an AI value of each animalwas calculated by following the formula discussed above. The average AIvalue of each group is provided in Table 17 and FIG. 13 . AlthoughCmyr-47 did not lower serum HDL-C in vivo, the average AI value ofanimals treated with Cmyr-47 was significantly lower than the averagevalue of the model controls, indicating that Cmyr-47 may be protectiveagainst atherosclerosis and other vascular disease includingcardiovascular diseases that are caused by accumulation of fat.

TABLE 17 Effect of Cmyr-47 on Serum HDL-C and AI (4 weeks post thetreatment) HDL-C Group Dosage/injection n (mmol/L) Al Normal control 10mL/kg PBS 10 2.29 ± 0.27 0.92 ± 0.11  Model control 10 mL/kg PBS 8  3.44± 0.19^(##) 4.75 ± 1.59^(##)  Positive Control 50 mg/kg FENO 8  3.13 ±0.30** 1.36 ± 0.38** Low-dose 10 mg/kg Cmyr-47 8 3.45 ± 0.22 3.62 ±1.49*  High-dose 30 mg/kg Cmyr-47 8 3.41 ± 0.18 2.39 ± 0.55** Note:compared with normal control, ^(#)P < 0.05, ^(##)P < 0.01; compared withmodel control, *P < 0.05, **P < 0.01

Example 3.6. Effect of Cmyr-47 on Serum Total Bile Acid (TBA)

Table 18 and FIG. 14 provide the measurements of serum TBA levels. Asexpected, the serum TBA levels in the model controls were higher thanthe normal controls. As compared with the model control group, the serumTBA levels of animals treated with Cmyr-47 were further elevated in adose-dependent fashion, confirming that Cmyr-47 is capable of inhibitingbile acid uptake in vivo. Measurements at week 4 confirmed that the highdose of Cmyr-47 significantly increased the serum TBA levels (P valuesless than 0.05). CsA and the low dose of Cmyr-47 also moderatelyelevated the serum TBA levels after 4 weeks of treatment, though thesignificance was not reached.

TABLE 18 Effect of Cmyr-47 on Serum TBA (mmol/L, χ ± S) TG level afterweeks of dosing Group Dosage/injection n Before dosing 2 w 4 w Normalcontrol 10 mL/kg PBS 10 24.61 ± 3.69 23.14 ± 2.65  18.13 ± 3.81  Modelcontrol 10 mL/kg PBS 8 36.19 ± 7.00  39.35 ± 12.04^(##)  41.63 ±18.61^(##) Positive Control 50 mg/kg FENO 8 35.74 ± 4.47 25.74 ± 4.25*24.45 ± 2.52* Low-dose 10 mg/kg Cmyr-47 8 37.28 ± 7.55 39.42 ± 11.1853.94 ± 32.15 High-dose 30 mg/kg Cmyr-47 8 32.15 ± 7.82 40.43 ± 5.96 64.08 ± 5.47* CsA treatment  5 mg/kg CSA 8 34.55 ± 6.67 41.38 ± 11.8352.73 ± 9.83  Note: compared with normal control, ^(#)P < 0.05, ^(##)P <0.01; compared with model control, *P < 0.05, **P < 0.01

Example 3.7. Effect of Cmyr-47 on Serum Glucose (GLU)

As shown in Table 19, hyperlipidemic animals did not show anysignificant hyperglycemic phenotype as compared with the normal controls(all values of P>0.05). The high and low doses of Cmyr-47 did not reducethe serum GLU levels below the normal glycemia displayed in the normalcontrols and the model controls (all values of P>0.05).

TABLE 19 Effect of Cmyr-47 on Serum GLU in Golden Hamster (mmol/L, χ ±SEM) GLU level after weeks of dosing Group Dosage/injection n Beforedosing 2 w 4 w Normal control 10 mL/kg PBS 10 4.09 ± 0.3 4.63 ± 0.8 4.99± 1.4 Model control 10 mL/kg PBS 8 3.99 ± 0.4 4.52 ± 0.9 4.34 ± 0.7Low-dose 10 mg/kg Cmyr-47 8 4.18 ± 0.3 3.87 ± 0.7 4.55 ± 1.0 High-dose30 mg/kg Cmyr-47 8 4.11 ± 0.5 4.10 ± 0.7 4.38 ± 0.4 Note: compared withnormal control group, ^(#)P < 0.05, ^(##)P < 0.01; compared with modelcontrol group, *P < 0.05, **P < 0.01

As discussed above, Cmyr-47 was capable of reversing hyperlipidemicphenotype in golden hamsters fed with a high-fat diet. In particular,the high dose of Cmyr-47 was capable of lowering all biologicalindicators measured in this study. In contrast, CsA, a bile acid uptakeinhibitor, failed to produce a similar effect. Furthermore, despite theeffective inhibition of bile acid uptake demonstrated by CsA in vitro,CsA treatment in vivo caused an increase of the serum TG and TC levels,confirming the hyperlipidemic effect of CsA.

Consistent with the effects on serum TC and TG levels of Cmyr-47, the AIvalues of Cmyr-47 treated animals were significantly lower than that ofthe non-treated hyperlipidemic animals, further demonstrating theefficacy of Cmyr-47 as a preventive medicine in cardiovascular diseases.In addition, other polypeptides derived from HBV showed similar efficacyin lowering serum TG, TC, and LDL-C, indicating that those polypeptidesare also capable of reversing hyperlipidemic phenotype in vivo.

Example 3.8. Effects of Various Doses of Cmyr-47 on Serum TC and TG

The hyperlipidemic golden hamster model was established as described inExample 3.1. After 2 weeks of the high-fat diet treatment, thehyperlipidemic phenotype of hamsters was confirmed when the animals hadserum total cholesterol (TC) levels higher than 10 mmol/L. A total of 32hyperlipidemic hamsters were randomly stratified into 4 groups(N=8/group): a model control group (10 mL/kg PBS, subcutaneously (sc)),a low-dose treatment group (Cmyr-47, 1 mg/kg/day, sc), a middle-dosetreatment group (Cmyr-47, 3 mg/kg/day, sc) and a high-dose treatmentgroup (10 mg/kg/day Cmyr-47, sc). Eight hamsters fed with the normalchow diet were used as a normal control group (10 mL/kg PBS, sc).

By following the protocol described in Example 3.1, Cmyr-47 wasadministered for 4 consecutive weeks. During the experiment, all animalsin the model control group, low, middle and high dose groups were fedwith the high fat diet, while the normal control group was fed with thenormal chow diet. Cmyr-47 or PBS was subcutaneously injected to thehamsters twice a day (9:00-10:00 in the morning and 16:00-17:00 in theafternoon). Each morning, food and water consumption, feces, and animalgrooming were monitored. The serum TC and TG levels of all animals weremeasured on week 2 and week 4 of treatment as described in Example 3.1and the results are summarized in Tables 20 and 21. The data wasanalyzed as described in Example 3.1.

As shown in Table 20 and FIG. 15 , the serum TC levels in the modelcontrols were significantly higher than those in the normal controlsduring the experiment (all values of P<0.01). The treatment with Cmyr-47at a dose of 1 mg/kg or 3 mg/kg had no effect on serum TC. However,hyperlipidemic animals treated with 10 mg/kg Cmyr-47 for 4 weeks showedmoderately reduced levels of serum TC as compared with the model controlgroup (P<0.05).

TABLE 20 Effect of Cmyr-47 on Serum TC (mmol/L, χ ± S) TC level afterweeks of dosing Group Dosage/injection n Before dosing 2 w 4 w Normalcontrol 10 mL/kg PBS 8  2.21 ± 0.3  4.3 ± 0.3 4.25 ± 0.3 Model control10 mL/kg PBS 8   13.45 ± 1.6^(##)  19.47 ± 4.9^(##)  19.88 ± 3.9^(##)Low-dose  1 mg/kg Cmyr-47 8 13.42 ± 1.5 19.75 ± 2.5 20.88 ± 4.0 Middle-dose  3 mg/kg Cmyr-47 8 13.33 ± 1.4 18.88 ± 2.1 20.99 ± 6.9 High-dose 10 mg/kg Cmyr-47 8 13.34 ± 1.4 16.47 ± 5.1 16.21 ± 4.2* Note:compared with normal control group, ^(#)P < 0.05, ^(##)P < 0.01;compared with model control group, *P < 0.05, **P < 0.01

As shown in Table 21 and FIG. 16 , the serum TG levels in the modelcontrols were significantly higher than the normal controls throughoutthe experiment (all values of P<0.01). The treatment with Cmyr-47 atdose of 1 mg/kg or 3 mg/kg had no effect on serum TG. Hyperlipidemicanimals treated with 10 mg/kg Cmyr-47 for 4 weeks expressed moderatelylowered serum TG levels comparing to the model control group (P<0.05).These data suggest that administering a therapeutically effective amountof Cmyr-47 to reach serum concentrations of Cmyr-47 capable ofbidirectionally regulating NTCP-mediated bile acid uptake may bebeneficial for lowering serum TC and TG levels in hyperlipidemicpatients.

TABLE 21 Effect of Cmyr-47 on Serum TG (mmol/L, χ ± S) TG level afterweeks of dosing Group Dosage/injection n Before dosing 2 w 4 w Normalcontrol 10 mL/kg PBS 8 2.04 ± 0.8 1.46 ± 0.3 1.11 ± 0.2 Model control 10mL/kg PBS 8  5.28 ± 1.0^(##)  6.48 ± 1.6^(##)  7.92 ± 5.2^(##) Low-dose 1 mg/kg Cmyr-47 8 4.78 ± 0.8 7.11 ± 2.1 7.41 ± 2.1 Middle-dose  3 mg/kgCmyr-47 8 5.37 ± 1.3 7.21 ± 2.7 6.91 ± 1.9 High-dose 10 mg/kg Cmyr-47 85.52 ± 1.3 5.55 ± 2.4  4.44 ± 2.1* Note: compared with normal controlgroup, ^(#)P < 0.05, ^(##)P < 0.01; compared with model control group,*P < 0.05, **P < 0.01

Example 4 Example 4.1. Methods and Materials Used in Treatment of ZuckerDiabetic Fatty Rats with Cmyr-47

The efficacy of Cmyr-47 as an anti-diabetic, anti-hyperlipidemic, and/oranti-hypercholesterolemic agent was tested in Zucker Diabetic Fatty(ZDF) rats, a spontaneous type II diabetes animal model. Male 60-daysold ZDF rats (n=40) and Zucker Lean (ZL) rats (n=6) were purchased fromVital River Laboratory Animal Technology Co., Ltd. (SCXK (Beijing)2012-0001) with animal quality certification numbers 11400700109970 and11400700109972. All animals were housed at 23±1° C. with 50-70% humidityunder specific-pathogen-free (SPF) environment and under a 12 hourlight:dark cycle (150-200 Lx), in a noise-controlled room (<50 dB) atthe Zhejiang Traditional Chinese Medicine University Animal ExperimentalResearch Center [SYXK(Zhe) 2013-0184]. Animals had a free access tofiltered and sterilized water in autoclaved water bottle. ZDF rats werefed with Purina #5008 diet purchased from Specialty Feeds, Inc.(Memphis, TN; Purina 5008; Catalogue No. SF06-019). ZL rats were fedwith a normal chow diet (basic feed) sterilized by Co⁶⁰ gammairradiation. Two ZDF rats or ZL rats were housed in each cage and cagebedding was changed once every 2 days. The experimental rat breeding andall other operations were in accordance with the principle of 3R withhumane care.

After 2 weeks of Purina #5008 diet or the normal chow diet, all animalswere fasted for 10 h with access to water. Each animal was then weighedand tail-bled to collect 0.3 mL of blood. The blood samples were furtheranalyzed for glycated hemoglobin (HbA1c). Additional 0.5 mL of blood wascollected and centrifuged at 3,000 rpm for 10 min in order to separateserum. The HbA1c, serum glucose (GLU), and insulin levels in the animalswere measured by kits provided below. Hitachi 7020 automatic biochemicalanalyzer was used for all the measurements.

30 animals with the fasting serum GLU levels close to the average levelwere selected and randomly divided into 5 groups: a model control group(10 ml·kg⁻¹ PBS), a low-dose treatment group (10 mg·kg⁻¹ Cmyr-47), ahigh-dose treatment group (30 mg·kg⁻¹ Cmyr-47), a positive control group(300 mg·kg⁻¹ Metformin), and a CsA treatment group (20 mg·kg⁻¹ CSA) with6 animals per group. Six ZL rats were used as a normal control group (10ml·kg⁻¹ PBS). PBS or Cmyr-47 was injected to animals subcutaneouslytwice per day (at 09:00 and 17:00). Meformin (MET) and CsA solutionswere given via oral gavage twice per day in the morning and afternoon.The dosing was conducted for 4 weeks. During the experiment, ZDF ratswere fed with Purina #5008 diet while ZL rats were fed the normal chowdiet.

Cmyr-47 was synthesized as described above, purified as a white powderby Shanghai HEP Pharmaceutical Co., Ltd (Shanghai, China; Lot number14011801), and stored at −20° C. Cmyr-47 was weighed and dissolved inPBS right before use. MET was manufactured by Bristol-Myers Squibb Co.,Ltd. (Shanghai, China) with approval number H20023370 and lot number ofAAD7878. CsA (Sandimmune®) was purchased from Novartis.

At the second-week of dosing, the animals were fasted for 10 h with freeaccess to water, and 0.3 mL of blood from each animal was collected viatail vein bleeding for measuring serum GLU, total cholesterol (TC),triglycerides (TG) and blood urea nitrogen (BUN). At the fourth-week ofdosing, the animals were fasted for 10 h and the blood was thencollected for the measurement of serum GLU, TC, TG and BUN, HbA1c andinsulin. After the last dose of drugs, the animals were fasted for 12 hand then anesthetized via intraperitoneal injection of 3% sodiumpentobarbital and cervical dislocation. Heart, kidneys, scapular fat,and abdominal fat of each animal were dissected for visual observationand weighed to calculate heart, kidney, and total fat index.

Pentobarbital sodium (content ≥95.0%; Lot number 20130112) was purchasedfrom Merck. TC, TG, GLU and BUN kits were purchased from DiaSysDiagnostic Systems (Shanghai, China). HbA1c detection reagent waspurchased from Trinity Biotech Inc., Ireland. Insulin ELISA detectionkit (Lot number: 0469636-1) was purchased from Bertin Pharma (France).All measurements using the purchased kits were conducted following themanufacturers' protocols provided in the kits.

SQP electronic balance was purchased from Sartorius ScientificInstruments (Beijing) Co., Ltd. (Beijing, china). ILS-3750 autoclave waspurchased from Sanyo, Japan. RO-MB-50 ultrapure water system wasmanufactured by Hangzhou Yongjieda Cleaning Science and Technology, Co.,Ltd. (Zhejiang, China). KQ-300DE ultrasound was purchased from KunshanUltrasonic Instruments Co., Ltd. (Jiangsu, China). Hitachi 7020automatic biochemical analyzer was purchased from Hitachi, Japan. Hb9210glycated hemoglobin analyzer, Trinity Biotech Inc, Ireland.Multifunctional microplate reader was purchased from Thermo FisherScientific, Co. (MA, USA).

SPSS 19.0 software (SPSS, Chicago, IL) was used for statisticalanalysis. All data were presented as mean±standard error mean (χ±SEM).ANOVA variance analysis was used to evaluate the data from the testresults. LSD test was used for pairwise comparisons. Values of thestatistical analyses were rounded to 2 decimal places.

Example 4.2. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum Glucose (GLU)

Hyperglycemia (i.e., increased blood glucose) is one of the mostprominent symptoms of type II diabetes. As shown in Table 22 and FIG. 17, hyperglycemic phenotype of ZDF rats was confirmed by comparing theserum GLU levels of the model controls with the normal controls (allvalues of P<0.01). MET is a well-known anti-diabetic agent thateffectively lowers glycemia in diabetic patients. As expected, ZDF ratstreated with MET showed significantly decreased fasting serum GLU ascompared with the model controls. Cmyr-47 was also capable of loweringglycemia in ZDF rats. In particular, by 4 weeks of treatments, both lowand high doses of Cmyr-47 effectively reduced the serum GLU levels inthe animals. The effect of Cmyr-47 on glycemia was dose-dependent. Incontrast, CsA treatment significantly elevated the serum GLU levels inthe animals as compared with the model control.

TABLE 22 Effect of Cmyr-47 on Fasting Serum GLU in ZDF Rats (mmol/L, χ ±SEM) GLU level after weeks of dosing Group Dosage/injection 0 2 4 Normalcontrol  10 mL/kg PBS 6.91 ± 0.22  6.23 ± 0.27 6.51 ± 0.19  Modelcontrol  10 mL/kg PBS  9.68 ± 0.90^(#)  19.36 ± 3.35^(##) 27.03 ±1.07^(##)  Positive control 300 mg/kg Met 9.71 ± 0.96   7.98 ± 1.02** 8.35 ± 0.58** Cmyr-47Lo  10 mg/kg Cmyr-47 9.82 ± 1.19 14.74 ± 0.6819.16 ± 1.23** Cmyr-47Hi  30 mg/kg Cmyr-47 9.73 ± 1.01  9.50 ± 1.72*10.50 ± 2.34** CsA treatment  20 mg/kg CsA 9.76 ± 1.22 22.38 ± 3.0833.75 ± 6.68*  Note: compare to normal control group, ^(#)P < 0.05,^(##)P < 0.01; compare to model control group, *P < 0.05, **P < 0.01

Additional polypeptides derived from HBV listed in Table 1 were alsotested in order to analyze their effects on glycemia in vivo byfollowing the same protocol described above. All polypeptide wasadministrated at a dose molarly equivalent to 30 mg/kg/day of Cmyr-47.

As shown in FIGS. 18A and 18B, hyperglycemic phenotype of ZDF rats wasconfirmed. As observed in the study above, the treatment with METsignificantly decreased fasting serum GLU in vivo. After 4 weeks oftreatment with HBV-derived peptides (Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35,Cmyr-30, Cmyr-25, Cmyr-20, Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47,Cstea-47, Cchol-47, Amyr-47, Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47or Hmyr-47), the fasting serum GLU levels of ZDF rats decreased ascompared with the model controls, indicating the efficacy of thepolypeptides as an anti-diabetic agent.

Example 4.3. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on HbA1c

HbA1c refers to glycated hemoglobin, and its level significantlyincreases when a subject is experiencing chronic hyperglycemia. As shownin Table 23 and FIG. 19 , consistent with hyperglycemic phenotype of ZDFrats, the HbA1c levels in the model controls were significantly higherthan the normal control throughout the study (all values of P<0.01).Also, consistent with the effect of MET on glycemia, the treatment withMET significantly decreased the HbA1c levels in ZDF rats (P<0.01). ZDFrats treated with Cmyr-47 also showed significantly reduced HbA1c levelsas compared with the model controls in a dose-dependent fashion,confirming the efficacy of Cmyr-47 as an anti-diabetic agent thatregulates glycemia in vivo. Consistent with the effect of CsA on theserum GLU levels, CsA treatment significantly increased the serum HbA1cafter 4 weeks of treatment.

TABLE 23 Effect of Cmyr-47 on HbA1c Level in ZDF Rats (%, χ ± SEM) HbA1clevel after weeks of dosing Group Dosage/injection 0 4 Normal control 10 mL/kg PBS 4.28 ± 0.02 4.55 ± 0.02  Model control  10 mL/kg PBS  6.27± 0.25^(##) 9.33 ± 0.40^(##)  Positive control 300 mg/kg Met 6.25 ± 0.315.07 ± 0.23** Cmvr-47Lo  10 mg/kg Cmyr-47 6.27 ± 0.24 6.82 ± 0.44**Cmyr-47Hi  30 mg/kg Cmyr-47 6.25 ± 0.31 5.73 ± 0.39** CsA treatment  20mg/kg CsA 6.23 ± 0.44 11.60 ± 1.03**  Note: compare to normal control,^(#)P < 0.05, ^(##)P < 0.01; compare to model control, *P < 0.05, **P <0.01

Additional polypeptides derived from HBV listed in Table 1 also showed asimilar effect on HbA1c when tested in the same animal model asdescribed above. As shown in FIGS. 20A and 20B, the HbA1c levels of themodel controls were higher than that of the ZDF rats treated with thepolypeptides. These results demonstrate that the polypeptides derivedfrom HBV are capable of effectively lowering glycemia and HbA1c, andconfirm the hyperglycemic effect of CsA.

Example 4.4. Effect of Cmyr-47 on Serum Insulin

Type II diabetic patients experience insulin resistance, overproductionof insulin, and ultimately insulin depravation due to pancreatic damage.To confirm that Cmyr-47 is capable of modulating insulin by preventingpancreatic damage, the insulin levels of animals treated with Cmyr-47were compared with animals treated with PBS or MET. As shown in Table 24and FIG. 21 , the initial serum insulin levels of ZDF rats weresignificantly higher than the normal controls. While the insulin levelsof ZDF rats remained higher than the normal controls at week 4, theabsolute levels significantly dropped as compared with the initiallevels. This change indicates that insulin resistance in ZDF ratsprogressed during the study, resulting in pancreatic failure. Thetreatment with MET appeared to be protective against the loss of seruminsulin, although the difference between the fasting insulin levels ofthe model controls and positive controls was not statisticallysignificant (P>0.05). Cmyr-47 treatment, however, completely stabilizedinsulin secretion, if not increased, at both doses, indicated by thesignificant difference between the model controls and the Cmyr-47treated animals (all values of P<0.05). These results demonstrate thatCmyr-47 may prevent pancreatic damage and therefore help a diabeticpatient to maintain proper insulin secretion.

TABLE 24 Effect of Cmyr-47 on Serum Insulin in ZDF Rats (ng/mL, χ ± SEM)Insulin level after weeks of dosing Group Dosage/injection 0 4 Normalcontrol  10 mL/kg PBS 0.46 ± 0.06 0.84 ± 0.06  Model control  10 mL/kgPBS  7.15 ± 1.38^(##)  3.85 ± 1.25^(##) Positive control 300 mg/kg Met6.74 ± 0.62 8.36 ± 3.68  Cmyr-47Lo  10 mg/kg Cmyr-47 7.99 ± 1.17 8.57 ±2.18* Cmyr-47Hi  30 mg/kg Cmyr-47 8.55 ± 1.07 9.56 ± 4.26* Note:compared with normal control group, ^(#)P < 0.05, ^(##)P < 0.01;compared with model control group, *P < 0.05, **P < 0.01

Example 4.5. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum Total Cholesterol (TC)

As discussed above, diabetic phenotypes include lipid dysregulationincluding elevated cholesterol and triglycerides in the blood stream. Asshown in Table 25 and FIG. 22 , the serum TC levels in the modelcontrols were consistently elevated as compared with the normal controls(all values of P<0.05). In contrast to the effect of MET on glycemia,however, MET appeared to be completely ineffective in lowering the serumTC levels in ZDF rats. CsA was also ineffective in lowering the serum TClevels in ZDF rats and by 4 weeks of treatment, CsA significantlyincreased the serum TC levels as compared with the model control.Notably, the high dose of Cmyr-47 significantly reduced the serum TClevels in ZDF rats as compared with the model controls, confirming thatCmyr-47 can regulate a wide array of biomarkers that are severelyelevated in diabetic patients.

TABLE 25 Effect of Cmyr-47 on Serum TC in ZDF Rats (mmol/mL, χ ± SEM) TClevel after weeks of dosing Group Dosage/injection 0 2 4 Normal control 10 mL/kg PBS 2.85 ± 0.03 3.18 ± 0.06 3.22 ± 0.04 Model control  10mL/kg PBS  3.68 ± 0.46^(#)  4.95 ± 0.15^(##)  6.95 ± 0.21^(##) Positivecontrol 300 mg/kg Met 3.82 ± 0.11 5.24 ± 0.35 6.95 ± 0.54 Cmyr-47Lo  10mg/kg Cmyr-47 3.72 ± 0.15 4.97 ± 0.19 6.63 ± 0.18 Cmyr-47Hi  30 mg/kgCmyr-47 3.60 ± 0.18  4.25 ± 0.27*  5.52 ± 0.41** CsA treatment  20 mg/kgCsA 3.65 ± 0.48  6.54 ± 0.63**  8.33 ± 0.65** Note: compared with normalcontrol group, ^(#)P < 0.05, ^(##)P < 0.01; compared with model controlgroup, *P < 0.05, **P < 0.01

Animals treated with additional polypeptides derived from HBV alsoshowed that those polypeptides are capable of reducing serum TC in vivo.As shown in FIGS. 23A and 23B, after 4 weeks of treatment withHBV-derived peptides (Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35, Cmyr-30,Cmyr-25, Cmyr-20, Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47, Cstea-47,Cchol-47, Amyr-47, Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47 orHmyr-47), the serum TC levels of ZDF rats were lower than that of themodel controls. These results demonstrate that while MET may target onlyone particular symptom, a polypeptide derived from HBV is capable oftargeting multiple pathways and therefore beneficial to managediabetes-related symptoms simultaneously.

Example 4.6. Effect of Cmyr-47 and Additional Polypeptides Derived fromHBV on Serum Triglycerides (TG)

In addition to serum TC, serum TG is also a biomarker for hyperlipidemiathat may be caused by diabetes. As expected, Table 26 and FIG. 24 showthat the serum TG levels in the model controls were significantly higherthan that of the normal controls throughout the study (all values ofP<0.01). Of note, MET significantly elevated the serum TG levels in ZDFrats at week 2 (P<0.01). While the difference was not significant, theserum TC levels in ZDF rats treated with MET remained higher than themodel controls at week 4. Consistent with the effect on serum TC levels,CsA further increased the serum TG levels in ZDF rats. In contrast, bothdoses of Cmyr-47 were capable of lowering the serum TG levels of ZDFrats and the effect of Cmyr-47 was dose-dependent.

TABLE 26 Effect of Cmyr-47 on Serum TG in ZDF Rats (mmol/mL, χ± SEM)Dosage/ TG level after weeks of dosing Group injection 0 2 4 Normal  10mL/ 0.58 ± 0.03   0.96 ± 0.08    1.47 ± 0.15   control kg PBS Model  10mL/ 9.80 ± 2.95^(##) 12.23 ± 1.36^(##)  13.43 ± 2.21^(##)  control kgPBS Positive 300 mg/ 8.32 ± 0.65  17.10 ± 2.02**  14.74 ± 1.99   controlkg Met Cmyr-  10 mg/kg 9.78 ± 0.48   9.50 ± 0.58    8.62 ± 0.41*   47LoCmyr-47 Cmyr-  30 mg/kg 8.11 ± 0.72   6.42 ± 0.92**   6.26 ± 0.64** 47Hi Cmyr-47 CsA  20 mg/kg 9.04 ± 0.84  14.34 ± 1.8**   19.03 ± 2.65** treatment CsA Note: compared with normal control, ^(#)P <0.5, ^(##)P<0.01; compared with model control, *P <0.05, **P <0.01

Additional polypeptides derived from HBV were also capable of reducingserum TG in vivo when tested in the same animal model described above.As shown in FIGS. 25A and 25B, the serum TG levels of the model controlsremained higher than ZDF rats treated with HBV-derived peptides(Cmyr-60, Cmyr-55, Cmyr-40, Cmyr-35, Cmyr-30, Cmyr-25, Cmyr-20,Cmyr-47+(−10), Cmyr-47+(−9), Cplam-47, Cstea-47, Cchol-47, Amyr-47,Bmyr-47, Dmyr-47, Emyr-47, Fmyr-47, Gmyr-47 or Hmyr-47), furtherconfirming that polypeptides derived from HBV are capable of regulatingglucose metabolism and lipid metabolism simultaneously.

Example 4.7. Effect of Cmyr-47 on Serum Blood Urea Nitrogen (BUN)

Elevated serum BUN reflects impaired renal function, which often occursin human diabetic patients. In ZDF model, diabetic phenotypes lead torenal dysfunction and the correlative elevation of serum BUN. Asexpected, Table 27 and FIG. 26 show that the serum BUN levels in themodel controls were significantly higher than that of the normalcontrols throughout the study. The treatment of MET was not capable ofreversing renal dysfunction as no significant BUN difference was foundbetween the model controls and the positive controls. However, the highdose of Cmyr-47 significantly lowered the serum BUN levels in ZDF ratsat week 4 (P<0.01), indicating that Cmyr-47 may protect a diabeticsubject against kidney damage and renal dysfunction.

TABLE 27 Effect of Cmyr-47 on Serum BUN in ZDF Rats (mmol/mL, χ ± SEM)BUN level after weeks of dosing Group Dosage/injection 0 2 4 Normalcontrol  10 mL/kg PBS 4.71 ± 0.22 5.54 ± 0.06 4.48 ± 0.09 Model control 10 mL/kg PBS  6.13 ± 0.51# 6.62 ± 0.35  7.96 ± 0.18## Positive control300 mg/kg Met 5.52 ± 0.21 6.58 ± 0.52 7.09 ± 0.36 Cmyr-47Lo  10 mg/kgCmyr-47 5.26 ± 0.29 6.83 ± 0.48 7.93 ± 0.38 Cmyr-47Hi  30 mg/kg Cmyr-475.83 ± 0.37 6.44 ± 0.45  6.65 ± 0.20** Note: compared with normalcontrol group, #P < 0.05, ##P < 0.01; compared with model control group,*P < 0.05, **P < 0.01

Example 4.8. Effect of Cmyr-47 on Organ Index

Diabetic patients have an increased risk of developing cardiovasculardiseases, renal dysfunction, and obesity. As discussed above, variousindexes such as heart index (HI), kidney index (KI), and total fat index(TFI) can be used as a quantitative indicator of the risk. As shown inTable 28 and FIG. 27A, the HI values of the model controls were higherthan the normal controls. The treatment with MET had no effect on the HIvalues. Surprisingly, both doses of Cmyr-47 significantly decreased theHI values of ZDF rats, indicating that Cmyr-47 can be beneficial toprevent adverse cardiovascular events.

As shown in Table 28 and FIG. 27B, the KI values of the model controlswere significantly higher than the normal controls (P<0.01), confirmingthat ZDF rats developed renal dysfunction during the study. Consistentwith the effect of Cmyr-47 on serum BUN, Cmyr-47 treatment also loweredthe KI values in ZDF rats, although the difference did not meet thestatistical significance.

Table 28 and FIG. 27C show that the TFI values of the model controlswere significantly higher than normal controls (P<0.01), indicating thatZDF rats reached morbid obesity. Although not significant, ZDF ratstreated with Cmyr-47 showed a trend of TFI values lower than the modelcontrols. Of note, MET did not show the similar trend and rather, itappeared to increase the TFI values in ZDF rats.

TABLE 28 Effect of Cmyr-47 on Organ Index in ZDF Rats After 4 Weeks ofTreatment (g/kg, χ ± SEM) Group Dosage/injection Heart index Kidneyindex Total fat index Normal control  10 mL/kg PBS 3.48 ± 0.09 6.64 ±0.13 27.47 ± 1.63 Model control  10 mL/kg PBS 3.63 ± 0.19  8.39 ±0.41^(#)  93.70 ± 5.01^(##) Positive control 300 mg/kg Met 3.63 ± 0.247.26 ± 0.55 94.64 ± 3.79 Cmyr-47Lo  10 mg/kg Cmyr-47  2.90 ± 0.13* 7.86± 0.63 89.18 ± 4.95 Cmyr-47Hi  30 mg/kg Cmyr-47  3.05 ± 0.10* 7.36 ±0.44 86.61 ± 5.03 Note: compared with normal control, ^(#)P < 0.05,^(##)P < 0.01; compared with model control, *P < 0.05, **P < 0.01

Example 4.9. Effect of Cmyr-47 on Serum Total Bile Acid (TBA)

To confirm that Cmyr-47 is capable of regulating the serum TBA level,the serum TBA level of each animal was measured. As shown in Table 29and FIG. 28 , the serum TBA levels in the model controls were higherthan the normal controls. As compared with the model control group, theserum TBA levels of ZDF rats treated with Cmyr-47 were further elevatedin a dose-dependent fashion. Measurements at week 4 confirmed that thehigh dose of Cmyr-47 significantly increased the serum TBA levels (Pvalues less than 0.05). Also, CsA significantly elevated the serum TBAlevels after 4 weeks of treatment (P values less than 0.01).

TABLE 29 Effect of Cmyr-47 on Serum TBA in ZDF Rats (mmol/mL, χ ± SEM)TBA level after weeks of dosing Group Dosage/injection 0 2 4 Normalcontrol  10 mL/kg PBS 42.61 ± 10.94 44.52 ± 8.62   28.51 ± 8.13 Modelcontrol  10 mL/kg PBS 55.51 ± 36.96 76.24 ± 31.22  59.15 ± 22.38#Positive control 300 mg/kg Met 32.83 ± 12.29 82.48 ± 27.02  82.97 ±15.82* Cmyr-47Lo  10 mg/kg Cmyr-47 37.51 ± 13.17 74.04 ± 33.35  74.38 ±28.53 Crayr-47Hi  30 mg/kg Cmyr-47 44.09 ± 12.20 85.47 ± 24.03 114.58 ±45.31* CsA treatment  20 mg/kg CsA 40.29 ± 6.69  78.53 ± 7.38  117.35 ±9.33** Note: compared with normal control group, #P < 0.05, ^(##)P <0.01; compared with model control group, *P < 0 05, **P < 0.01

MET is a widely used antidiabetic drug that can efficiently modulateglycemia in vivo. As presented above, the efficacy of MET in glycemiaregulation was also confirmed in this study. However, MET failed toprovide benefits against lipid dysregulation and associated diseases, asevident by the results that MET was not capable of lowering serum TC,TG, and BUN levels, and HI values in ZDF rats. As shown inhyperlipidemic animal models, CsA treatment not only increased the serumTG and TC levels, but further increased the serum GLU levels, suggestingthat effective inhibition of bile acid uptake in vitro does not alwaystranslate to a therapeutic effect on lipid and glucose metabolism invivo. In contrast, Cmyr-47 performed well all across the tests. Asdemonstrated above, Cmyr-47 was capable of modulating glycemia and lipidmetabolism while providing protection against pancreas damage, renaldysfunction, and cardiovascular diseases. Thus, Cmyr-47 performedsuperior as an anti-hyperglycemia, anti-hypercholesterolemia,anti-hyperlipidemia, and anti-adiposity agent over MET and showed acomprehensive effect on multiple diabetic phenotypes simultaneously.

1. A method of treating or preventing a metabolic disease, acardiovascular disease, a heart diseases, or a kidney impairment in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a polypeptide or a pharmaceuticalcomposition comprising the polypeptide to produce a serum concentrationof the administered polypeptide at a concentration above a concentrationthreshold of 93 nmol/L, wherein the polypeptide comprises an amino acidsequence derived from Hepatitis B virus (HBV); wherein the metabolicdisease chosen from diabetes; hyperglycemia; hypoglycemia;hyperinsulinemia; hyperlipidemia; hypertriglyceridemia;hypercholesterolemia; heart disease; metabolic syndrome; atheroscleroticdisease; coronary heart disease; coronary artery disease; peripheralarterial disease; angina pectoris; cerebrovascular disease; acutecoronary syndrome; myocardial infarction; stroke; cardiovasculardisease; Alzheimer's disease; dyslipidemias; familial combinedhyperlipidemia; familial hypertriglyceridemia; familialhypercholesterolemia; heterozygous hypercholesterolemia; homozygoushypercholesterolemia; familial defective apolipoprotein B-100; polygenichypercholesterolemia; remnant removal disease; hepatic lipasedeficiency; dyslipidemia caused by dietary indiscretion, hypothyroidism,drugs including estrogen and progestin therapy, beta-blockers, andthiazide diuretics; nephrotic syndrome; chronic renal failure; Cushing'ssyndrome; primary biliary cirrhosis; glycogen storage disease; hepatoma;cholestasis; acromegaly; insulinoma; isolated growth hormone deficiency;kidney impairment; obesity; and alcohol-induced hypertriglyceridemia;wherein the cardiovascular disease is an atherosclerotic disease.
 2. Themethod of claim 1, wherein the metabolic disease involves dysregulationof lipid metabolism and/or dysregulation of glucose metabolism.
 3. Themethod of claim 1, wherein the polypeptide is capable of reducing orstabilizing the level or activity of one or more chemical or biologicalmolecules associated with metabolism in the subject, wherein thechemical or biological molecule is chosen from glucose, cholesterol,triglycerides, free fatty acids, amino acids, hormones, LDL-C, HDL-C,HbA1c, blood urea nitrogen, and minerals; or the polypeptide is capableof lowering the level of total cholesterol, triglycerides, and/or LDL-Cin the subject.
 4. The method of claim 1, wherein the polypeptide iscapable of lowering the level of glucose and/or HbA1c in the subject, orthe polypeptide is capable of stabilizing the level of insulin in thesubject.
 5. The method of claim 1, wherein the polypeptide is capable ofreducing or stabilizing the level or value of one or more physiologicalparameters that measure metabolic changes chosen from glycemia, bloodpressure, body weight, fat mass, body mass index (BMI), inflammation,atherosclerosis index, heart index, kidney index, total fat index, andhomeostatic model assessment (HOMA) index.
 6. The method of claim 1,wherein the polypeptide comprises an amino acid sequence of the pre-S1region of HBV genotype A, B, C, D, E, F, G, or H.
 7. The method of claim1, wherein the polypeptide comprises the sequence of amino acids 13-59of the pre-S1 region of HBV genotype C.
 8. The method of claim 1,wherein the polypeptide comprises an amino acid sequence selected fromSEQ ID NOs: 21-40, or has at least about 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99%% identity to an amino acid sequenceselected from SEQ ID NOs: 21-40.
 9. The method of claim 6, wherein thepolypeptide comprises at the N-terminus and/or the C-terminus a nativeflanking amino acid sequence from the pre-S1 region of HBV.
 10. Themethod of claim 9, wherein the native flanking amino acid sequence fromthe pre-S1 region of HBV has 1-10, 1-8, 1-5, or 1-3 amino acids inlength.
 11. The method of claim 1, wherein the polypeptide comprises theglycine corresponding to amino acid 13 of the pre-S1 region of HBVgenotype C, and or the asparagine corresponding to amino acid 20 of thepre-S1 region of HBV genotype C.
 12. The method of claim 1, wherein thepolypeptide comprises an N-terminal modification with a hydrophobicgroup, and/or a C-terminal modification that is capable of stabilizingthe polypeptide.
 13. The method of claim 12, wherein the hydrophobicgroup is chosen from myristic acid, palmitic acid, stearic acid, oleicacid, linoleic acid, cholesterol, and arachidonic acid; and theC-terminal modification is amidation (amination) orisopentanediolization.
 14. The method of claim 1, wherein thepolypeptide comprises SEQ ID NO: 23, and wherein the polypeptide furthercomprises an N-terminal modification with myristic acid and a C-terminalmodification with amination; or wherein the polypeptide comprises SEQ IDNO:
 3. 15. The method of claim 1, wherein the polypeptide is capable ofbinding to sodium taurocholate cotransporting polypeptide (NTCP). 16.The method of claim 1, wherein the serum concentration of theadministered polypeptide in the subject is above 500 ng/ml.
 17. Themethod of claim 1, wherein the polypeptide or the pharmaceuticalcomposition comprising the polypeptide is administered to the subjectbefore, concurrently with, or after the administration of atherapeutically effective amount of at least one second agent.
 18. Themethod of claim 17, wherein the second agent is chosen from anantihyperlipidemic agent, an antihyperglycemic agent, an antidiabeticagent, an antiobesity agent, and a bile acid analogue.
 19. The method ofclaim 18, wherein the second agent is chosen from insulin, metformin,sitagliptin, colesevelam, glipizide, simvastatin, atorvastatin,ezetimibe, fenofibrate, nicotinic acid, orlistat, lorcaserin,phentermine, topiramate, obeticholic acid, and ursodeoxycholic acid. 20.The method of claim 1, wherein the polypeptide or the pharmaceuticalcomposition comprising the polypeptide is administered to the subject byat least one mode chosen from parenteral, intrapulmonary, intranasal,intralesional, intramuscular, intravenous, intraarterial,intraperitoneal, and subcutaneous administration.